Preparation of fagopyritols and uses therefor

ABSTRACT

The present invention describes isolated Fagopyritol A1, isolated Fagopyritol A3, and isolated Fagopyritol B3. Compositions which include two or more of Fagopyritol A1, Fagopyritol A2, Fagopyritol A3, Fagopyritol B1, Fagopyritol B2, Fagopyritol B3, and D-chiro-inositol, at least one of which is an isolated Fagopyritol A1, isolated Fagopyritol A3, or isolated Fagopyritol B3, are also disclosed. Methods for preparing substantially pure Fagopyritol A1, Fagopyritol A3, Fagopyritol B3, or mixtures thereof from buckwheat are also described. The fagopyritols can be used to prepare pharmaceutical compositions, the administration of which can be used to treat diabetes.

This application is a continuation-in-part of U.S. Patent Application Ser. No. 09/073,467, filed May 6, 1998 now U.S. Pat. No. 6,162,795, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/045,927, filed May 7, 1997, which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to fagopyritols and to methods for using fagopyritols.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a major global health problem which is recognized by the World Health Organization to be reaching epidemic proportions. It is now the fourth leading cause of death in most developed countries and a disease that is increasing rapidly in countries undergoing industrialization. Estimates of worldwide diabetes prevalence have increased from 30 million in 1985 to more than 100 million in 1994. Diabetes mellitus is a disease caused by defective carbohydrate metabolism and characterized by abnormally large amounts of glucose in the blood and urine. Diabetes mellitus can eventually damage the eyes, kidneys, heart, and limbs, and can endanger pregnancy.

Diabetes mellitus is usually classified into two types. Type I, or insulin-dependent diabetes mellitus (“IDDM”), formerly called juvenile-onset diabetes because it occurs primarily in children and young adults, has been implicated as one of the autoimmune diseases. Rapid in onset and progress, it accounts for about 10 to 15 percent of all cases. Type II, or non-insulin-dependent diabetes mellitus (“NIDDM”), formerly called adult-onset diabetes, is usually found in persons over 40 years old and progresses slowly. Often it is not accompanied by clinical illness in its initial stages and is detected instead by elevated blood or urine glucose levels.

Diabetes is considered a group of disorders with multiple causes, rather than a single disorder. The human pancreas secretes a hormone called insulin that facilitates the entry of glucose into tissues of the body and its utilization, thus providing energy for bodily activities. In a person with diabetes, however, the entry of glucose is impaired, a result either of a deficiency in the amount of insulin produced or of altered target cells. Consequently, sugar builds up in the blood and is excreted in the urine. In the Type I diabetic, the problem is almost always a severe or total reduction in insulin production. In the Type II diabetic, the pancreas often makes a considerable quantity of insulin, but the hormone is unable to promote the utilization of glucose by tissues.

With adequate treatment most diabetics maintain blood-sugar levels within a normal or nearly normal range. This permits them to live normal lives and prevents some long-term consequences of the disease. For the Type I diabetic with little or no insulin production, therapy involves insulin injections. For Type II diabetics, most of whom are at least moderately overweight, therapy is based on diet control, weight reduction, and exercise. Weight reduction appears partially to reverse the condition of insulin resistance in the tissues. If a Type II patient's blood-sugar level is still high, the physician may add insulin injections to the treatment regimen. In many cases, the need for insulin injections is not due to a deficiency in insulin but, instead, due to the patient's reduced ability to utilize insulin efficiently because of a deficiency of galactosamine D-chiro-inositol, an insulin mediator.

Besides the discomfort associated with its administration by injection, the problem of controlling the dose of insulin also exists. The hypoglycemia produced by an insulin overdose may lead to tremors, cold sweat, piloerection, hypothermia, and headache, accompanied by confusion, hallucinations, bizarre behavior, and, ultimately, convulsions and coma.

Therefore, it would be advantageous to control a diabetic's blood-sugar level without resort to insulin injections. The present invention is directed to providing such control.

SUMMARY OF THE INVENTION

The present invention relates to an isolated Fagopyritol A1, an isolated Fagopyritol A3, and an isolated Fagopyritol B3.

The present invention is also directed to a composition comprising two or more of Fagopyritol A1, Fagopyritol A3, Fagopyritol A3, Fagopyritol B1, Fagopyritol B2, Fagopyritol B3, and D-chiro-inositol. The composition comprises at least one isolated Fagopyritol A1, isolated Fagopyritol A3, or isolated Fagopyritol B3.

The present invention also relates to a substantially pure fagopyritol selected from the group consisting of Fagopyritol A1, Fagopyritol A3, and Fagopyritol B3.

In another aspect, the present invention relates to a method for preparing a material selected from the group consisting of Fagopyritol A1, Fagopyritol A3, Fagopyritol B3, and a mixture thereof. Buckwheat is contacted with a solvent under conditions effective to produce a crude extract. The crude extract contains non-fagopyritol materials and one or more fagopyritols selected from the group consisting of Fagopyritol A1, Fagopyritol A3, and Fagopyritol B3. The non-fagopyritol materials are then separated from the one or more fagopyritols.

The isolated fagopyritols of the present invention can be used in a pharmaceutical composition which also includes a pharmaceutical carrier. This pharmaceutical composition or, alternatively, the substantially pure fagopyritols of the present invention or the isolated fagopyritols of the present invention can be administered to a patient to treat diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are schematic diagrams showing the components of a buckwheat seed. FIG. 1A is a vertical section through a mature buckwheat achene. FIG. 1B is a horizontal cross section of a mature buckwheat achene. FIG. 1C is a horizontal cross section of a mature buckwheat groat (dehulled buckwheat achene). FIG. 1D is a horizontal cross section of a mature buckwheat groat showing its milling fractions and fracture planes.

FIG. 2 is a gas chromatogram of trimethylsilylated soluble components extracted from embryo tissues of mature buckwheat seed with ethanol:water (1:1, v:v). The peaks are labeled as follows: (a) D-chiro-inositol, (b) myo-inositol, (c) phenyl α-D-glucoside (internal standard), (d) sucrose, (e) Fagopyritol A1, (f) Fagopyritol B1, (g) galactinol, (h) raffinose, (i) unknown, (j) unknown, (k) Fagopyritol A2, (l) Fagopyritol B2, (m) unknown (probably digalactosyl myo-inositol), (n) stachyose, and (o) Fagopyritol A3.

FIGS. 3A-3C are gas chromatograms of trimethylsilylated soluble components extracted from embryo tissues of 20 DAP buckwheat seed with ethanol:water (1:1, v:v). FIG. 3A is a gas chromatogram before hydrolysis;

FIG. 3B is a gas chromatogram after hydrolysis with α-galactosidase enzyme for 23 h; and FIG. 3C is a gas chromatogram after acid hydrolysis with 2N trifluoroacetic acid (“TFA”) at 70° C. for 3 h. The peaks are identified as follows: (a) D-chiro-inositol, (b) myo-inositol, (c) phenyl α-D-glucoside (internal standard), (d) sucrose, (e) Fagopyritol A1, (f) Fagopyritol B1, (g) galactinol, (k) Fagopyritol A2, (1) Fagopyritol B2, (1) galactose, (2) glucose, and (3) fructose. Trimethylsilyl (“TMS”) derivatives of glucose, fructose, and galactose capture the anomeric forms of the sugars as distinct TMS products.

FIG. 4 is a graph showing the fractionation of fagopyritols from buckwheat seed. Mature groats were extracted with 50% ethanol, heated at 80° C. for 45 min to inactivate hydrolytic enzymes and passed through a 10,000 M_(r) cutoff filter, and the soluble fraction was evaporated to dryness. The residue was dissolved in 5 ml of acetonitrile:water (75:25, v:v) and chromatographed on a 9×290 mm column filled with 3-aminopropyl bonded silica gel, 9% functionalized. Components were eluted at 0.4 ml/min gravity flow with acetonitrile:water (75:25, v:v) and chromatographed on a 9×290 mm column filled with 3-aminopropyl bonded silica gel, 9% functionalized. Components were eluted at 0.4 ml/min gravity flow with acetonitrile:water (75:25, v:v, for fractions 1-70; and 60:40 acetonitrile:water for fractions 71-90). One ml of each 5-ml fraction was evaporated to dryness, derivatized with trimethylsilylimidazole (“TMSI”), and analyzed by gas chromatography. A 1-ml sample of selected fractions was evaporated to dryness for enzymatic and/or acid hydrolysis. Components in each fraction are identified as follows: (a) chiro-inositol plus myo-inositol plus sucrose, (b) Fagopyritol A1, (c) unknown, (d) Fagopyritol B1, (e) Fagopyritol A2, and (f) Fagopyritol B2.

FIG. 5 shows structural formulas for the galactinol series. The enzyme galactinol synthase transfers a α-galactosyl from UDP-galactose to the 3-position of D-myo-inositol to form galactinol (O-α-D-galactopyranosyl-(1→3)-D-myo-inositol). Galactinol serves as a galactose donor to form the raffinose series oligosaccharides in many seeds. Galactinol can also serve as acceptor to form digalactosyl myo-inositol (O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→3)-D-myo-inositol) found in seeds of vetch (Vicia saliva L.) and other legumes (Petek et al., “Isolation of Two Galactosides of myo-Inositol from Vetch Seeds,” C.R. Acad. Sci. (Paris) Serie D: Sci. Nat., 263:195-197 (1966) (“Petek I”), and Petek et al., “Purification and Properties of α-Galactosidase in Germinating Vicia sativa seeds,” Eur. J Biochem., 8:395-402 (1969) (“Petek II”), which are hereby incorporated by reference). Small quantities of higher oligomers (trigalactosyl myo-inositol and tetragalactosyl myo-inositol) may occur in legume seeds (Courtois et al., “Distribution of Monosaccharides, Oligosaccharides and Polyols,” in Harborne, et al. eds., Chernotaxonomy of the Leguminosae, New York:Academic Press, Inc., pp. 207-229 (1971) (“Courtois”), which is hereby incorporated by reference).

FIG. 6 is a graph showing the germination and desiccation tolerance of dehulled ‘Mancan’ buckwheat seeds harvested at 6 to 32 days after pollination (“DAP”) and germinated fresh (o) or after rapid drying at 12% relative humidity (“RH”) over a saturated solution of LiCl (). Germination was recorded at 10 d at 25° C. on wet paper towels.

FIGS. 7A-7E are graphs showing the slow-desiccation induction tolerance in immature (10 DAP) dehulled seeds of ‘Mancan’ buckwheat by incubating at 93% RH during d 1, 87% RH during d 2, 75% RH during d 3, 51% RH during d 4,45% RH during d 5, and 33% RH during d 6. Fresh seeds and seeds after incubation for 1 to 6 d at the successive lower RH environments (over saturated solutions of various salts) were rapidly dried at 12% RH and 22° C. for I week. After desiccation, seeds were germinated on wet paper towels at 25° C. Seeds that germinated with 7 d and had normal radicle growth were considered to be desiccation tolerant. FIG. 7A shows the desiccation tolerance () and d to 50% germination (∘) as a function of d during slow drying. FIG. 7B shows amount of cyclitols D-chiro-inositol () and myo-inositol (▪) per embryo as a function of d during slow drying. FIG. 7C shows the amount of galactosyl cyclitols Fagopyritol B1 (), Fagopyritol A1 (filled triangle), and galactinol (▪) per embryo as a function of d during slow drying. FIG. 7D shows the amount of digalactosyl cyclitols Fagopyritol B2 (), Fagopyritol A2 (filled triangle), and digalactosyl myo-inositol (□) per embryo as a function of d during slow drying. FIG. 7E shows the amount of sucrose () as a function of d during slow drying.

FIGS. 8A-8E are graphs showing various properties as a function of d at high relative humidity (98%) incubation of fresh 10 DAP immature dehulled ‘Mancan’ buckwheat seeds. FIG. 8A shows the desiccation tolerance () and d to 50% germination (∘) as a function of d at high relative humidity. FIG. 8B shows the amount of cyclitols D-chiro-inositol () and myo-inositol (▪) per embryo as a function of d at high relative humidity. FIG. 8C shows the amount of galactosyl cyclitols Fagopyritol B1(), Fagopyritol A1 (filled triangle), and galactinol (▪) per embryo as a function of d at high relative humidity. FIG. 8D shows the amount of digalactosyl and trigalactosyl cyclitols Fagopyritol A2 (filled triangle), Fagopyritol B2 (), Fagopyritol A3 (∘), and digalactosyl myo-inositol (□) per embryo as a function of d at high relative humidity. FIG. 8E shows the amount of sucrose () per embryo as a function of d at high relative humidity.

FIGS. 9A-9J are graphs showing the loss of desiccation tolerance and changes in saccharides and cyclitols during 0 to 48 h of germination of dehulled mature ‘Mancan’ buckwheat seeds at 25° C. on wet paper towels. FIG. 9A shows the desiccation tolerance () and onset of germination (∘) as a function of h of germination on wet paper towels at 25° C. FIG. 9B shows the axis length () as a function of h of germination on wet paper towels at 25° C. FIG. 9C shows the amount of cyclitols D-chiro-inositol () and myo-inositol (∘) per axis as a function of h of germination on wet paper towels at 25° C. FIG. 9D shows the amount of cyclitols D-chiro-inositol () and myo-inositol (∘) per cotyledon as a function of h of germination on wet paper towels at 25° C. FIG. 9E shows the amount of Fagopyritol B1 (), Fagopyritol A1 (∘), Fagopyritol B2 (▪), and Fagopyritol A2 (□) per axis as a function of h of germination on wet paper towels at 25° C. FIG. 9F shows the amount of Fagopyritol B1 (e), Fagopyritol A1 (∘), Fagopyritol B2 (▪), and Fagopyritol A2 (□) per cotyledon as a function of h of germination on wet paper towels at 25° C. FIG. 9G shows the amount of sucrose () per axis as a function of h of germination on wet paper towels at 25° C. FIG. 9H shows the amount of sucrose () per cotyledon as a function of h of germination on wet paper towels at 25° C. FIG. 9I shows the amount of glucose (), fructose (∘), and unknown (▪) per axis as a function of h of germination on wet paper towels at 25° C. FIG. 9J shows the amount of maltose (),maltotriose (∘), and maltotetraose (▪) per cotyledon as a function of h of germination on wet paper towels at 25° C.

FIG. 10 is a graph of the amount of various fagopyritols present in various fractions separated on a 3-aminopropyl-functionalized silica gel column. The material loaded on the column was the enriched Fagopyritol A3 fraction collected from a P2 column.

FIG. 11 is a graph of the amount of various fagopyritols present in various fractions separated on a 3-aminopropyl-functionalized silica gel column. The material loaded on the column was selected fractions from a P2 column.

FIG. 12 is a graph of the amount of various fagopyritols present in various fractions separated on a 3-aminopropyl-functionalized silica gel column. The material loaded on the column was the extract from 50 g of whole groat flour (Birkett Mills).

FIG. 13 is a graph of the amount of various fagopyritols present in various fractions separated on a 3-aminopropyl-functionalized silica gel column. The material loaded on the column was the extract from 50 g of whole groat flour (Birkett Mills).

FIG. 14 is a graph of the amount of various fagopyritols present in various fractions separated on a 3-aminopropyl-functionalized silica gel column. The material loaded on the column was the extract from 50 g of whole groat flour (Birkett Mills).

FIG. 15 is a graph of the amount of various fagopyritols present in various fractions separated on a 3-aminopropyl-functionalized silica gel column. The material loaded on the column was the extract from 50 g of light buckwheat flour (Minn-Dak).

FIG. 16 shows carbon-Celite column (cc38) chromatography of a pilot plant extract (20 g total sugars) containing fagopyritols eluted stepwise with ethanol: water (v/v).

FIG. 17 shows expanded scale plots from FIG. 16 illustrating the elution of di-galactosyl myo-inositol (DGMI), Fagopyritol A2 (A2), Fagopyritol B3 (B3), tri-galactosyl myo-inositol (T, TGMI), and Fagopyritol A3 (A3).

FIG. 18 is a gas chromatogram illustrating Fagopyritol A3 (A3, 38.23 min), Fagopyritol B3 (B3, 39.00 min), and tri-galactosyl myo-inositol (TGMI, 39.46 min) plotted with expanded scale to illustrate peaks of interest.

FIG. 19 shows three superimposed traces of GC chromatograms illustrating separation of peaks for fractions enriched in Fagopyritol A3 (A3; 37.83 min), Fagopyritol B3 (B3; 38.50 min), and tri-galactosyl myo-inositol (TGMI; 38.97 min), respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to fagopyritols. Fagopyritol is a general term used herein to mean an unspecified (α-galactosyl D-chiro-inositol or its salt or derivative. More particularly, the present invention relates to isolated Fagopyritol A1, particularly Fagopyritol A1s have the following Formula I:

and salts thereof.

The present invention further relates to an isolated Fagopyritol A3, particularly Fagopyritol A3s having the following formula II:

The present invention further relates to an isolated Fagopyritol B3, particularly Fagopyritol B3s have the following Formula III:

and salts and derivatives thereof.

Salts of the Fagopyritol A1s, A3s, and B3s of the present invention can be the reaction product of a base having a pKa (i.e., -log Ka) greater than the pKa of one or more of the fagopyritols' hydroxyl groups, such as a metal hydroxide or alkoxide, an amonium hydroxide, or an amine (e.g. a tertiary amine, like triethyl amine). Exemplary salts are alkali metal salts, such as lithium salts, sodium salts, and potassium salts, alkali earth metal salts, such as calcium salts and barium salts, ammonium salts, sufonium salts, and phosphonium salts.

Derivatives of the Fagopyritol A1s, A3s, and B3s of the present invention include, for example, the reaction products of the fagopyritols with compounds bearing a carbon having a positive charge, such as an alkyl halide, in which case the derivative is an ether of the fagopyritol, or a carboxylic acid halide (e.g., acetyl chloride) or anhydride (e.g., acetic anhydride), in which case the derivative is an ester of the fagopyritol (e.g., the acetate).

As used herein, an isolated fagopyritol is one which is substantially free of other buckwheat components with which it naturally occurs. It is to be understood that the isolated fagopyritols of the present invention can be prepared by a variety of methods including, for example, by extraction from buckwheat or other natural sources, as well as by chemical synthesis.

The present invention also relates to substantially pure Fagopyritol A1, a substantially pure Fagopyritol A3, and substantially pure Fagopyritol B3, preferably having Formula I, II, and III, respectively, their salts and derivatives. The present invention also relates to substantially pure Fagopyritol A2, particularly Fagopyritol A2s have the following Formula IV:

and salts thereof.

The present invention further relates to substantially pure Fagopyritol B1, particularly fagopyritols having the following Formula V:

and their salts and derivatives. The present invention also relates to substantially pure Fagopyritol B2, particularly fagopyritols having the following Formula VI:

as well as their salts and derivatives. As referred to herein, substantially pure means substantially free of other compounds or materials, such as galactinol, myo-inositol, digalactosyl myo-inositol, phytin, aromatic materials (e.g. polyphenols and pigments and other colored aromatic materials), cell wall particles, proteins, and acids (e.g. organic acids, nucleic acids, and amino acids) and their salts. Typically, substantially pure fagopyritols are those having greater than about 95% purity, such as greater than about 98% purity or from about 95% to about 99% purity.

As indicated above, the fagopyritols of the present invention are not limited by their method of preparation. One particularly preferred method, involves purification of the subject fagopyritols from buckwheat. Briefly, buckwheat is contacted with a solvent under conditions effective to produce a crude extract containing non-fagopyritol materials and one or more fagopyritols selected from the group consisting of Fagopyritol A1, Fagopyritol A3, and Fagopyritol B3. The one or more fagopyritols are then separated from the non-fagopyritol materials. This process is described in greater detail below.

Any portion of the buckwheat plant (Fagopyrum esculentum) can be used in preparing the fagopyritols of the present invention.

The structure of developing buckwheat seeds has been reviewed and illustrated by Obendorf et al., “Structure and Chemical Composition of Developing Buckwheat Seed,” pp. 244-251 in Janick et al., eds., New Crops, New York:John Wiley & Sons (1993) (“Obendorf”), which is hereby incorporated by reference. Photographs of mature buckwheat seeds have been illustrated in reviews by Marshall et al., “Buckwheat: Description, Breeding, Production, and Utilization,” Advances in Cereal Science and Technology, 5:157-210 (1982) (“Marshall”) and Pomeranz, “Buckwheat: Structure, Composition, and Utilization,” CRC Critical Reviews in Food Science and Nutrition, 19:213-258 (1983), which are hereby incorporated by reference. Most of the structural information in the latter reviews originated from a study of the scanning electron microscopy of the buckwheat kernel by Pomeranz et al., “Scanning Electron Microscopy of the Buckwheat Kernel,” Cereal Chemistry, 49:23-25 (1972), which is hereby incorporated by reference. The embryo is positioned with the radicle (root) of the embryo at the pointed top of the buckwheat achene (the name of the mature buckwheat fruit) as shown schematically in FIG. 1A. As described by Obendorf, which is hereby incorporated by reference, the cotyledons have extended through the soft (mostly liquid) endosperm to reach the bottom of the endosperm by 8 days after pollination. The achene is attached to the raceme (name of inflorescence or flower cluster) of the mother plant by the pedicel. The embryo, including the axis and cotyledons continues to grow through the endosperm, curling around the margins of the endosperm and reaching the maximum dry weight at 16 days after pollination. The endosperm continues to accumulate dry matter (mostly starch) until 24 to 28 days after pollination. After a few more days the buckwheat grain (achene or fruit) is mature and dry.

A horizontal cross-section view (see line marked “xs view” in FIG. 1A) of the mature buckwheat achene is schematically illustrated in FIG. 1B. This “top-down” view illustrates the hull (pericarp, fruit coat) as the outer layer of the achene (black with light cross-hatch markings in FIGS. 1B and 1C). The hull is a hard fibrous structure that is usually black or dark brown in color. The hull is the first layer to be removed during milling to produce dehulled groats (the true “seed” without the pericarp) (FIG. 1C). The outer part of the dehulled groat is the “skin” or seed coat (also called testa). This is a fibrous layer composed of seed coat cells with thickened cell walls and remnants of the nucellus (perisperm; 1-3 cells thick). The outermost layer of cells in the endosperm is called the aleurone layer and usually adheres to the seed coat and nucellus tissues in mature grains. The aleurone is a single layer of small cells that have thicker cell walls than those of the central endosperm cells, do not contain starch, and remain “alive” upon drying of the seed. The remainder of endosperm cells have thinner cell walls and are packed with starch granules as illustrated in Marshall, which is hereby incorporated by reference.

The buckwheat embryo has an axis (circle in center) and two thin, leaf-like cotyledons that wind through the endosperm to the margin and then bend to continue along the outer part of the endosperm near the seed coat (FIGS. 1B and 1C; embryo structures in black, endosperm structures in white, and seed coat or “skin” in shaded gray). In the mature grain, the cotyledons nearly surround the endosperm. The embryo (axis plus cotyledons) remain alive in the mature dry grain. After germination, the embryo forms the buckwheat seedling. In mature dry grain, the embryo and aleurone layer cells contain all of the fagopyritols and D-chiro-inositol in the buckwheat grain. The embryo (as well as the aleurone layer) contains most of the protein and nearly all of the oil, phytin, minerals, vitamins, and rutin, but only traces of starch. Nearly all of the starch is in the endosperm, but there is no starch in the aleurone layer, the outer most layer of the endosperm.

Whole groat flour contains the entire groat (FIG. 1C) ground without separation of fractions. Because of the fibrous nature of the seed coat, larger fragments usually form during milling of buckwheat groats. The fracture plane is usually inside the layers with thick-walled cells. Likewise in wheat, oats, and barley dry milling, the bran fraction contains the pericarp (with seed coat), nucellar remnants, aleurone, and subaleurone layers. The fracture plane is between the thicker-walled subaleurone layer and the thinner-walled central endosperm in these grains. Similarly, the aleurone layer of buckwheat usually adheres to the “bran” or “skins” fraction during milling. In the bran fraction, the “seed coat” (also known as testa), or “skin” adheres to the outer of the two cotyledons, and that part of the cotyledon tears off and separates with the bran fraction (see schematic illustration of theoretical fracture planes in FIG. 1D). Not all skins have adhering cotyledon tissues. Most of the endosperm has soft thin-walled cells that form flour during milling. In some seeds, the cotyledons in the center of the endosperm are quite loose and may flake off in larger segments of “pure” cotyledon tissue. These larger fragments of the cotyledons and axis may separate into the bran fraction also, but much of the soft embryo tissue is pulverized and separates with the light flour fraction that is rich in starch. For these reasons, the bran fractions can be rich sources of fagopyritols and D-chiro-inositol. Likewise, the light flour fraction contains fagopyritols from embryo fragments embedded in the endosperm, but the concentration is reduced due to dilution with large amounts of starch. Since the embryo matures before the endosperm, embryo size varies less than endosperm size in mature grain. In smaller seeds (less endosperm), the cotyledons are in contact with more of the seed coat while in larger seeds (more endosperm) the cotyledons are in contact with less of the total seed coats. “Purified bran” without hulls in the fraction, are rich in fagopyritols, minerals, protein, and oil.

Important to this description is that the embryo cells (cotyledons and axis) and the cells of the aleurone layer are the cells that contain sucrose, fagopyritols, rutin, phytin, most of the minerals, most of the vitamins, nearly all of the oil, and a high concentration of protein. Cells of the embryo and aleurone layer are “living” cells in the dry seed, but cells of the subaleurone, central endosperm, and seedcoat (testa or skin) are dead. Cells of the hull (pericarp) also are dead in a dry seed. Thus, any fraction that is rich in embryo and aleurone layer cells will be rich in these components. The central endosperm cells are predominantly starch, and, therefore, the light flour fraction is rich in starch. However, because the embryo traverses the central endosperm, much of the embryo breaks into small fragments and ends up in the light flour fraction, but more would be in the dark flour fraction (includes the skins, too) or in whole groat flour.

Preferred portions of the buckwheat plant for use in preparing the fagopyritols of the present invention are the buckwheat embryo, especially the embryonic axis, or any milling fraction rich in embryo, cotyledon, axis, or aleurone layer cells including “bran” or “purified bran” milling fractions. Because of its ready availability, it has been found that buckwheat flour, such as commercial fancy buckwheat flour (preferably having about 12.3% moisture) or whole groat flour prepared from commercial whole white groats, is an excellent source of buckwheat for the isolation of fagopyritols. Alternatively, the flour can be produced from buckwheat seeds, preferably dehulled buckwheat seeds. Irrespective of the buckwheat source, extraction is improved when the buckwheat, such as buckwheat flour, is ground to a fine powder, preferably to a powder which passes through a 100-mesh sieve. The buckwheat flour can be raw or defatted, defatting being effected by mixing the raw buckwheat with a solvent for fatty acids, such as alkane solvents, preferably hexanes. Although defatting increases the efficiency of the initial extraction steps, subsequent filtration and chromatography are generally more difficult when employing defatted flour.

The flour is then extracted with a suitable solvent. Suitable solvents for the extraction process include water, alcohol, and water/alcohol mixtures. Water extracts typically contain more suspended materials, probably because of the higher solubility of proteins and starch in water. Alcohol and alcohol/water extracts generally contain less suspended materials and also minimize microbial and endogenous enzyme activities during extraction. Suitable alcohols for use in alcohol and alcohol/water extraction include methanol, ethanol, isopropanol, and n-propanol, ethanol being preferred. Water/ethanol mixtures are the preferred extraction solvents. Of these, the preferred solvent mixtures are those where the ethanol-to-water volume ratio is from about 1:2 to about 2:1, more preferably about 1:1.

Extraction is carried out by mixing a suitable quantity of the extraction solvent with the buckwheat flour or other buckwheat source, preferably with agitation. Agitation can be effected with any suitable means, such as with a blender, preferably having a “high shear” polytron head, a paddle mixer, or a shearing mixer. When a blender is used as the agitation apparatus, good results are obtained when the blender is cycled 10 seconds on and then 10 seconds off for 1 or 2 min. The volume of the extraction solvent used per unit weight of flour is not particularly critical to the practice of the present invention. Typically, solvent/flour ratios (ml/g) of from about 1:1 to about 200:1 are suitable, with solvent/flour ratios of from about 10:1 to about 50:1 being preferred, and solvent/flour ratios of about 20:1 being most preferred.

The temperature at which the extraction is carried out is not particularly critical to the practice of the present invention. However, after hot extraction, the mixture can be too thick (due to swelling of starch present in the buckwheat) to filter or centrifuge efficiently, resulting in low volumes of extracted fagopyritols. Cold extraction at temperatures from about 4° C. to about room temperature (approximately 25° C.), preferably from about 10C to about room temperature, and most preferably at about room temperature generally produces the highest yields.

The duration of the extraction process depends on a number of factors, including the type and vigorousness of agitation, the shape of the container in which extraction is carried out, the solvent-to-flour ratio, the temperature at which the extraction process is carried out, the volume of the material being extracted, the fineness of the grinding (particle size) of the material being extracted, and the like. Preferably, extraction is carried out for a period of time sufficient to produce a homogeneous material. Typically, about 15 seconds to about 1 hour, preferably from about 1 to about 5 minutes, more preferably from about 1 to about 2 minutes is effective.

The yield of extracted fagopyritols can frequently be increased by repeating the extraction process one, two, or more times. Typically, on the first extraction, between 85 and 95% of the fagopyritols present are extracted. The second extraction, when performed, yields an additional 8-10%, and the third extraction generally produces yet an additional 1%.

After the first and optional additional extraction steps, the homogenate is permitted to settle, and the solvent is removed. Although solvent removal can be achieved by any suitable means, filtration, particularly ultrafiltration, or centrifugation yields the best results. The crude extract (e.g., the supernatant after centrifugation or the filtrate after filtration) is then preferably heated to a temperature sufficient to inactivate any alpha-galactosidase and/or other enzymes present in the buckwheat that may hydrolyze the fagopyritols. Generally, deactivation can be effected by heating the crude extract to a temperature of from about 70 to about 90° C., preferably about 80° C. After cooling, the crude extract is preferably clarified by filtration, concentrated by evaporation, and then further clarified by ultrafiltration. The composition of the crude extract can be monitored during the extraction process and subsequent filtration, centrifugation, deactivation, evaporation, or clarification, by standard methods, including gas-liquid chromatography, high performance liquid chromatography (“HPLC”), spectrophotometric analysis, dye-binding protein assays, and atomic absorption analysis. If necessary, the enriched fraction of fagopyritols can be fractionated by ethanol precipitation to remove unwanted soluble carbohydrates of higher oligomeric forms (e.g., pentasaccharides and higher).

Recovery of the fagopyritols can be further enhanced by removing cell wall particles and large proteins, for example, by passing the crude extract through a filter having a cut off of from about 5000 M_(r) to about 20,000 M_(r), preferably about 10,000 M_(r).

Additionally or alternatively, the fagopyritols can be further purified by removing non-carbohydrate contaminants with, for example, food-grade ion exchange resins, such as Syburn Resin (available from Aftech, Inc., Rochester, N.Y.), polyvinylpolypyrrolidone, (“PVPP”) (e.g., Polyclar-VT, available from ISP Technologies, Inc., Wayne, N.J.), bentonite (e.g., KWK Bentonite agglomerated powder, available from Presque Isle Wine Cellars, North East, Penn.), and diatomaceous earth powder (available, e.g., from Celite Corporation, Lamoc, Calif.). Illustratively, charged materials can be removed from the crude extract, by passing the crude extract through an ion exchange material, such as Amberlite ERA-94 ion exchange resin and Dowex 50W×4 ion exchange resin (both available commercially from Sigma Chemical Company, St. Louis). Further purification of the fagopyritols can be effected by removing aromatic materials, such as polyphenolic materials, aromatic pigments, and other brown-colored contaminants, by passing the crude extract through a column containing a material which absorbs aromatic materials, such as polyvinylpyrrolidone (“PVP”) or activated charcoal. The fagopyritols can then be precipitated by chilling the concentrated crude extract or by adding an alcohol, such as ethanol, to the concentrated extract, or both.

The resulting mixture of fagopyritols and other sugars can then be separated into its component fagopyritols, for example, chromatographically. For example, chromatographic separation can be carried out on an amine functionalized silica gel or by using a carbon-celite column or both. Generally, the carbon-celite column is better at removing water-soluble low-molecular-weight colored contaminants.

The preferred amine functionalized silica gel, 3-aminopropyl-functionalized silica gel, is available commercially from Sigma-Aldrich (Milwaukee, Wis.). The degree of functionalization of the silica gel is not critical to the practice of the present invention. Preferably, this silica gel is from about 5% to about 15% functionalized, and, more preferably, it is about 9% functionalized. Suitable solvents for loading the crude extract on and eluting the various fagopyritols from the 3-aminopropyl-functionalized silica gel can be readily ascertained by those skilled in the art. Preferably, the separation is carried out using an acetonitrile/water eluent. More preferably, the crude extract is loaded as an acetonitrile:water (70:30, v:v) solution and is eluted, stepwise, with 70:30 (v:v) acetonitrile:water, 60:40 (v:v) acetonitrile:water, and then 50:50 (v:v) acetonitrile:water. Subsequent to elution, the 3-aminopropyl functionalized silica gel can be regenerated by with 60:40 (v:v) acetonitrile:water. Generally, the order of elution when using a 3-aminopropyl-functionalized silica gel is as follows: mono saccharides, D-chiro-inositol, myo-inositol, sucrose, trehalose, Fagopyritol A1, Fagopritol B1, galactinol, Fagopyritol A2, Fagopritol B2, di-galactosyl myo-inositol, Fagopyritol A3, Fagopyritol B3, and tri-galactosyl myo-inositol.

When a celite-carbon column is employed to effect chromatographic separation, suitable columns include those which contain a mixture of carbon (e.g., Darco-G60, J. T. Baker, Phillipsburg, N.J. and celite (e.g., Celite 545-AW, Spelco, Bellefonte, Penn.). Generally, the column material is prepared by slurrying the carbon and celite in distilled water. The slurry is then packed into a suitably sized column, and the packed column is then washed with distilled water. The crude extract can be loaded using any suitable solvent, water being preferred. The carbon-celite column can be eluted with, for example, water, alcohol (e.g., ethanol), or combinations thereof. Illustratively, elution can be carried out using 50% ethanol (i.e., a 50:50 (v/v) mixture of ethanol and water). Alteratively, the elution can be carried out stepwise, first eluting with distilled water, and then eluting with a series of mixtures of ethanol and water having increasing ethanol content. For example, the column can be first eluted with distilled water, then with 5% ethanol, then with 10% ethanol, then with 20% ethanol, then with 30% ethanol, then with 40% ethanol, and then with 50% ethanol. In some cases, one or more of the elution steps can be omitted. For example, elution can proceed directly from the 20% ethanol elution to the 50% elution without conducting the 30% and 40% ethanol elutions. Alternatively, elution can be carried out using a water/ethanol gradient solvent system. Generally, the order of elution when using carbon-celite is as follows: D-chiro-inositol, myo-inositol, monosaccharides, Fagopyritol B1, galactinol, Fagopyritol A1, trehalose, Fagopyritol B2, sucrose, di-galactosyl myo-inositol, Fagopyritol A2, Fagopyritol B3, tri-galactosyl myo-inositol and Fagopyritol A3.

The composition of various fractions can be analyzed by evaporating aliquots from the fractions to dryness, derivitizing with trimethylsilylimidazole (“TMSI”) in pyridine (1:1) to produce trimethylsilyl (“TMS”) derivatized carbohydrates, and analyzing the TMS-derivatized carbohydrates by, for example, high resolution gas chromatography. By pooling fractions containing one particular fagopyritol and evaporated to dryness, substantially pure fagopyritol is produced. Fractions containing mixtures of fagopyritols can be rechromatographed to effect their separation.

The present invention also relates to a composition which includes two or more of Fagopyritol A1, Fagopyritol A2, Fagopyritol A3, Fagopyritol B1, Fagopyritol B2, Fagopyritol B3, and D-chiro-inositol. Preferably, this composition includes at least one isolated Fagopyritol A1, Fagopyritol A3, or Fagopyritol B3. Illustrative compositions are those which include isolated Fagopyritol A1 and isolated Fagopyritol A2, Fagopyritol B2 and isolated Fagopyritol B3, D-chiro-inositol and isolated Fagopyritol B3, and the like. Preferably, the composition is substantially free of one or more of galactinol, myo-inositol, digalactosyl myo-inositol, phytin, aromatic materials (e.g. polyphenols and pigments and other colored aromatic materials), cell wall particles, proteins, and acids (e.g. organic acids, nucleic acids, and amino acids) and their salts. It was observed that a mixture of fagopyritols was degraded within 6 hours in the presence of human fecal bacteria under in vitro conditions in the laboratory. Therefore, it is believed that the fagopyritols are digested by bacteria in the digestive tract to release free D-chiro-inositol for uptake, or in the case of monomers or dimers, may be taken up by cells of the digestive tract.

The aforementioned fagopyritols and compositions are useful in treating diabetes in patients, such as mammals, including dogs, cats, rats, mice, and humans, by administering an effective amount of the above-described isolated fagopyritols, substantially pure fagopyritols, or compositions to such patients. The aforementioned fagopyritols and compositions may also be useful in treating polycystic ovary syndrome (see Nestler et al., New England J of Med., 340:1314-1320 (1999), which is hereby incorporated by reference). For example, the substantially pure fagopyritols, the compositions, or one or more of the isolated fagopyritols of the present invention can be administered alone, or the isolated fagopyritols of the present invention can be administered in combination with suitable pharmaceutical carriers or diluents. The diluent or carrier ingredients should be selected so that they do not diminish the therapeutic effects of the fagopyritols or compositions of the present invention. Suitable pharmaceutical compositions include those which include a pharmaceutical carrier and, for example, an isolated Fagopyritol A1, an isolated Fagopyritol A3, or an isolated Fagopyritol B3. The pharmaceutical composition can, additionally, contain Fagopyritol A2, Fagopyritol B1, Fagopyritol B2, or all three.

The fagopyritols and compositions herein can be made up in any suitable form appropriate for the desired use; e.g., oral, parenteral, or topical administration. Examples of parenteral administration are intraventricular, intracerebral, intramuscular, intravenous, intraperitoneal, rectal, and subcutaneous administration. The preferred route for administration is oral. In cases where the fagopyritols are administered topically or parenterally, it is preferred that the fagopyritols be pre-hydrolyzed.

Suitable dosage forms for oral use include tablets, dispersible powders, granules, capsules, suspensions, syrups, and elixirs. Inert diluents and carriers for tablets include, for example, calcium carbonate, sodium carbonate, lactose, and talc. Tablets may also contain granulating and disintegrating agents, such as starch and alginic acid; binding agents, such as starch, gelatin, and acacia; and lubricating agents, such as magnesium stearate, stearic acid, and talc. Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption. Inert diluents and carriers which may be used in capsules include, for example, calcium carbonate, calcium phosphate, and kaolin. Suspensions, syrups, and elixirs may contain conventional excipients, such as methyl cellulose, tragacanth, sodium alginate; wetting agents, such as lecithin and polyoxyethylene stearate; and preservatives, such as ethyl-p-hydroxybenzoate. Dosage forms suitable for parenteral administration include solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain suspending or dispersing agents known in the art.

For oral administration either solid or fluid unit dosage forms can be prepared. For preparing solid compositions, such as tablets, a suitable fagopyritol or composition, as disclosed above, is mixed with conventional ingredients, such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia methylcellulose, and functionally similar materials as pharmaceutical diluents or carriers. Capsules are prepared by mixing the disclosed fagopyritols or compositions with an inert pharmaceutical diluent and filling the fixture into a hard gelatin capsule of appropriate size. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the fagopyritol or composition with an acceptable vegetable oil, light liquid petrolatum, or other inert oil.

Fluid unit dosage forms for oral administration such as syrups, elixirs, and suspensions can be prepared. The water-soluble forms can be dissolved in an aqueous vehicle together with sugar, aromatic flavoring agents, and preservatives to form a syrup. An elixir is prepared by using a hydro-alcoholic (ethanol) vehicle with suitable sweeteners, such as sugar and saccharin, together with an aromatic flavoring agent. Suspensions can be prepared with a syrup vehicle with the aid of a suspending agent, such as acacia, tragacanth, methylcellulose, and the like.

When the fagopyritols or compositions are administered orally, suitable daily dosages can be based on suitable doses of free D-chiro-inositol, such as those described in U.S. Pat. No. 5,124,360 to Lamer et al., which is hereby incorporated by reference. It is believed that about half of the fagopyritols as extracted is D-chiro-inositol, mostly as bound D-chiro-inositol with small amounts of free D-chiro-inositol. Therefore, suitable doses of fagopyritol are about twice the suitable doses of D-chiro-inositol. Typically, for oral administration, suitable daily doses are from about 5 mg to about 200 mg of the fagopyritol or composition per kilogram of the subject's body weight.

Alternatively, the fagopyritols of the present invention can be administered orally in foodstuffs. For example, fagopyritols can be incorporated in purified form or in the form of buckwheat bran in bread, bread rolls, or other foodstuffs to form an edible product for consumption of fagopyritols. Fortification of breads, bread rolls, and other foodstuffs with extracted fagopyritols can provide a way to incorporate larger quantities of fagopyritols into a daily diet. Suitable procedures for bread preparation can be found, for example, in Brown, The Tassajara Bread Book, Boston: Shambhala Publications (1986), which is hereby incorporated by reference.

For parenteral administration, fluid unit dosage forms are prepared utilizing the aforementioned fagopyritols or compositions and a sterile vehicle, water being preferred. The fagopyritol or composition, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions, the fagopyritol or composition can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampule and sealing. Advantageously, adjuvants, such as a local anesthetic, preservative, and buffering agents, can be dissolved in the vehicle. To enhance the stability, the fluid unit dosage form can be frozen after filling into the vial, and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial, and an accompanying vial of water for injection is supplied to reconstitute the liquid prior to use. Parenteral suspensions are prepared in substantially the same manner except that the fagopyritol or composition is suspended in the vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration. The fagopyritol or composition can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the parenteral suspension to facilitate uniform distribution of the fagopyritol or composition. Parenteral dosages can range from about 5 mg to about 200 mg of fagopyritol or composition per kilogram of the subject's body weight per day. Preferably, the daily parenteral dosage would be considerably less than the dose per kilogram of subject body weight, considering that, in oral administration, the galactose from the fagopyritols would be consumed by microbes in the digestive tract whereas, in parenteral administration the galactose would contribute to blood sugar levels.

Alternatively, the fagopyritol or composition can be incorporated into a sustained release formulation and surgically implanted using conventional methods. Suitable sustained release matricies include those made of ethylene vinyl acetate and other bicompatible polymers.

For topical administration, carriers, such as phospholipid vesicles, which contain the aforementioned fagopyritols may facilitate uptake of fagopyritols through the skin.

As indicated above, it is believed that the fagopyritols are digested in the digestive tract by bacteria to release free D-chiro-inositol for uptake. It is known that D-chiro-inositol is an anti-oxidant and, more particularly, a hydroxyl radical scavenger. Accordingly, the fagopyritol and compositions of the present invention can also be used as a source of the antioxidant D-chiro-inositol, for example, by administering, preferably orally, the subject fagopyritols and compositions to a subject.

The present invention is further illustrated by the following examples.

EXAMPLES Example 1 Materials and Methods

Plant Materials. Embryos of common buckwheat (Fagopyrum esculentum Moench cv. ‘Mancan’, Polygonaceae) were analyzed for soluble carbohydrates and cyclitols during seed development and in mature seeds. Mature seeds of common buckwheat cv. ‘Manor’ and ‘Tartary’ buckwheat (Fagopyrum tartaricum (L.) Gaertn.) also were analyzed. Buckwheat plants were grown in a greenhouse at 24° C. day (14 h) and 18° C. night (10 h) as described previously (Horbowicz et al., “Changes in Sterols and Fatty Acids of Buckwheat Endosperm and Embryo During Seed Development,” J. Agric. Food Chem., 40:745-750 (1992), which is hereby incorporated by reference). Natural sunlight was supplemented 14 h daily with approximately 740 μmol m⁻² s⁻¹ incandescent light from 1000-watt Sylvania metal halide lamps. Embryos were removed from seed harvested at 6 to 32 DAP and at maturity. To evaluate the effect of temperature, 4-week-old plants from the greenhouse were transferred to growth chambers at 18 and 25° C. constant temperature until seed maturity. Plants received 16 h of fluorescent light daily at about 300 μmol m⁻² s⁻¹. Plants with pin-type flowers and plants with thrum-type flowers were pollinated by hand by legitimate cross pollination, pin x thrum and thrum x pin, between plants within the same temperature regime in growth chambers or greenhouse. Fresh and dry weight, water, and water concentration are expressed as mean ±SE of the mean for three replications of 6 to 10 seeds per treatment for pericarp, dehulled groat (seed), embryo, and endosperm on greenhouse-grown plants. For analysis of soluble components in axis and cotyledon tissues, three replications of 10 embryos each were divided into axis and cotyledon fractions. For analysis of endosperm and embryo fractions, three replications of three mature, dry, and dehulled (pericarp removed) seeds where separated by hand into embryo and endosperm fractions. The endosperm fraction contained seed coat but not pericarp. For comparison among cultivars and species, three replications of three dehulled groats each were analyzed.

Carbohydrate and Cyclitol Standards. Glucose, fructose, galactose, sorbitol, maltose, maltotriose, maltotetraose, myo-inositol, sucrose, raffinose, stachyose, phenyl α-D-glucoside (internal standard), quercetin, and rutin were purchased from Sigma Chemical Co. (St. Louis, Mo.). Galactinol was a gift from T. M. Kuo (Peoria, Ill.); D-ononitol, sequoyitol, and L-(+)-bomesitol were gifts from F. A. Loewus (Pullman, Wash.); D-chiro-inositol and D-pinitol were gifts from S. J. Angyal (Kennington, New South Wales Australia); O-α-D-galactopyranosyl-(1→2)-4-O-methyl-D-chiro-inositol (galactopinitol A), O-α-D-galactopyranosyl-(1→3)-3-O-methyl-D-chiro-inositol (galactopinitol B), D-pinitol, and D-chiro-inositol were gifts from J. G. Streeter (Wooster, Ohio); verbascose, D-chiro-inositol, L-chiro-inositol, and D-pinitol were gifts from P. Wiirsch (Lausanne, Switzerland); verbascose and D-chiro-inositol were gifts from P. Adams and R. G. Jensen (Tucson, Ariz.); and D-chiro-inositol and L-chiro-inositol were gifts from A. Richter (Vienna, Austria).

Extraction and Analysis of Soluble Carbohydrates. Axis and cotyledon tissues from three replicates of 7 embryos at 20 DAP or 5 to 10 embryos of developing (6 to 32 DAP) and mature, dry seeds were separated, weighed, and thoroughly homogenized in a mortar with 0.6 ml of ethanol:water (1:1 v:v) containing 100 μg of phenyl a-D-glucoside as internal standard. The homogenate and rinse were combined in a 1.5-ml microfuge tube, heated at 80° C. for 45 min to inactivate endogenous enzymes, and centrifuged in a Beckman Microfuge E at full speed for 20 min. The residue was re-extracted, and the combined supernatants were passed through a 10,000 M_(r) cutoff filter. Aliquots of the filtrate were transferred to silylation vials and evaporated to dryness in a stream of nitrogen gas. The residues were kept overnight in a desiccator over phosphorus pentoxide to remove traces of water. The dry residues were derivatized with 200 μl a of silylation mixture (TMSI:pyridine, 1:1, v:v) in tightly capped silylation vials (Supelco, Belfonte, Calif.) at 70° C. in an aluminum block heater for 30 min and then cooled to 22° C. One μl of the soluble carbohydrates derivatized by TMSI was injected into a split-mode injector of a Hewlett Packard 5890 Series II gas chromatograph equipped with a flame ionization detector and a Hewlett Packard 3396A Integrator. Soluble carbohydrates were analyzed on a DB-1 capillary column (15 in length, 0.25 mm ID, and 0.25 gm film thickness; J&W Scientific, Folsom, CA) operated with a programmed initial temperature of 150° C., adjusted to 200° C. at 3° C.Imin, adjusted to 325° C. at 7° C./min, and held at 325° C. for 20 min. The injector port was operated at 335° C., and the detector was operated at 350° C. The carrier gas was helium at 3.0 ml/min, split 1:50. The detector gas was hydrogen at 30 ml/min and air at 300 ml/min.

Quantities of soluble carbohydrates were determined by extrapolation from standard curves, and the ratios of the area of peaks for each known saccharide or cyclitol to the area of the peak for phenyl (-D-glucoside, the internal standard, were plotted against known amounts of each component. All standards were dissolved in ethanol:water (1:1, v:v) before analysis to capture the anomeric forms as TMS derivatives. Amounts of unknown carbohydrates were estimated by calculation with nearest known standard. Quantities are expressed as mean +SE of the mean for three replications of each treatment.

Fractionation ofFagopyritols ofBuckwheat Seeds. Soluble components extracted in ethanol:water (1:1, v:v) were evaporated to dryness. The residue was dissolved in 5 ml of acetonitrite:water (75:25, v:v) and chromatographed on a 9 x 290 mm column filled with 3-aminopropyl bonded silica gel, 9% fimctionalized (Aldrich Chemical Company, Inc., Milwaukee, Wisconsin). Components were eluted at 0.4 mvmin gravity flow with acetonitrile:water (75:25, v:v, for fractions <70; and 60:40, v:v, for fractions >70). One ml of each 5-ml fraction was evaporated to dryness and analyzed by gas chromatography as described above. A 1-ml sample of selected fractions was evaporated to dryness for enzymatic and/or acid hydrolysis.

Enzymatic Digestion. One ml of green coffee bean a.-galactosidase (EC 3.2.1.22) (Boehringer Mannheim Corporation, Indianapolis, IN) in 3.2 M ammonium sulfate was dialyzed overnight against 1000 ml of distilled water to remove the ammonium sulfate that interferes with the silylation reaction and causes variable results. Dialyzed enzyme in 0.1 ml of water was added to carbohydrate or cyclitol in 0.1 ml of water and incubated at 37° C. After 24 h, 0.2 ml of absolute ethanol and 100 jg of internal standard were added. The mixture was heated to 90° C. for 5 min to inactivate the enzyme and centrifuged through a 10,000 M_(r) cutoff filter. The filtrate was evaporated to dryness in a stream of nitrogen gas, silylated, and analyzed by gas chromatography as above.

Acid Hydrolysis. Dry samples of selected compounds were hydrolyzed with 2 N TFA for 3 h at 70° C. Compounds and hydrolysis products were identified by comparison to authentic standards by gas chromatography or by gas chromatography/mass spectroscopy (“CGC-MS”) and/or proton nuclear magnetic resonance (“IH-NMR”).

Desiccation Tolerance. Immature seeds were removed from plants at 6 to 32 DAP, dehulled (pericarp removed), and germinated fresh or tested for desiccation tolerance (Blackman et al., “Maturation Proteins and Sugars in Desiccation Tolerance of Developing Soybean Seeds,” Plant Physiol., 100:225-230 (1992) (“Blackman I”), which is hereby incorporated by reference). Dehulled groats were dried rapidly at 12% relative humidity (“RH”) over a saturated solution of LiCl at 25° C. After 6 d, seeds were humidified overnight at high relative humidity above water and germinated on wet paper towels at 25° C. Percent germination at 7 d and d-to-50%-germination are reported.

Immature seeds were harvested at 10 DAP, dehulled, and dried slowly during a 6 d period by transferring seeds daily to successively lower relative humidity (92, 87, 75, 51, 45, and 32% RH) maintained in sealed desiccators over saturated salt solutions of KNO₃, Na₂CO₃, NaCl, Mg(NO₃)₂, K₂CO₃, and MgCl₂, respectively (Blackman et al., “Maturation Proteins Associated with Desiccation Tolerance in Soybean,” Plant Physiol., 96:868-874 (1991) (“Blackman II”), which is hereby incorporated by reference). Dehulled groats (seeds) were tested for desiccation tolerance by transferring to 12% RH for 7 d and then testing the desiccated seeds (following humidification) for germination after 7 d on wet paper towels at 25° C. For the high-relative-humidity control treatment, immature seeds were placed in desiccators over water for 6 d (Blackman II, which is hereby incorporated by reference), before testing for desiccation tolerance. Embryos were removed after each step in the slow-drying process or after each day at high relative humidity and assayed for soluble carbohydrates and cyclitols.

Mature seeds of ‘Mancan’ commercial seedstock were germinated on wet paper towels for 0, 6, 12, 18, 24, 36, and 48 h and then rapidly dried at 12% RH over a saturated solution of LiCl as above. After 7 d of rehydration on wet paper towels at 25° C., seedlings with normal radicle growth were classified as desiccation tolerant. Axis or cotyledons were assayed for soluble carbohydrates and cyclitols.

Example 2 Oligosaccharides and Cyclitols in Embryos of Mature Seeds

A typical gas chromatogram of soluble sugars and cyclitols extracted from embryos of mature dry buckwheat seed from commercially available registered seedstocks of ‘Mancan’ is shown in FIG. 2. Buckwheat embryos are believed to be unique because they accumulate galactosyl cyclitols (sugar alcohols) instead of the raffinose series oligosaccharides. A major soluble component, named Fagopyritol B1 (see FIG. 2, peak f) after the species name Fagopyrum, accumulated to 40% of the total soluble sucrose and cyclitols in buckwheat embryos. Sucrose (FIG. 2, peak d) accounted for another 42% of the total soluble sugars and cyclitols.

Fagopyritol B1 is an α-galactosyl derivative of the cyclitol D-chiro-inositol, and has the same retention time as O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol, a minor component in soybean seeds (Schweizer et al., “Purification and Structure Determination of Three α-D-galactopyranosylcyclitols from Soya Beans,” Carbohydr. Res., 95:61-71 (1981) and Traitler et al., “Gas Chromatographic Separation of Sugars by On-Column Injection on Glass Capillary Column,” J. High Resolut. Chromatogr. Chromatogr. Commun., 7:558-562 (1984), which are hereby incorporated by reference). Evidence suggested that two different galactosyl chiro-inositol isomers, Fagopyritol A1 and Fagopyritol B1 (FIG. 2, peaks e and f), and two different digalactosyl chiro-inositol isomers, Fagopyritol A2 and Fagopyritol B2 (FIG. 2, peaks k and 1), were present in buckwheat embryos. In addition, small quantities of Fagopyritol A3 (FIG. 2, peak o), a trigalactosyl chiro-inositol, were detected in commercial seedstocks. At least three of the five were not reported previously. Two underivatized free cyclitols, D-chiro-inositol and myo-inositol (FIG. 2, peaks a and b), were found in buckwheat. In contrast to many legume seeds that accumulate pinitol and several galactosyl derivatives of pinitol including galactopinitol A, galactopinitol B, and ciceritol (Horbowicz et al., “Seed Desiccation Tolerance and Storability: Dependence on Flatulence-Producing Oligosaccharides and Cyclitols—Review and Survey,” Seed Sci. Res., 4:385-405 (1994) (“Horbowicz”), which is hereby incorporated by reference), buckwheat seeds did not contain pinitol or pinitol derivatives. Buckwheat accumulated only small amounts of galacitinol (O-α-D-galactopyranosyl-(1→3)-D-myo-inositol) (FIG. 2, peak g) or digalactosyl myo-inositol (O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→3)-D-myo-inositol) (FIG. 2, peak m) (Petek I and Courtois, which are hereby incorporated by reference). Unlike maize or soybean and other legumes, such as lupine (Horbowicz, which is hereby incorporated by reference), buckwheat embryos accumulated very little raffinose (O-α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside) (FIG. 2, peak h) and only small quantities of stachyose(O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside) (less than 1% of the total soluble components) (FIG. 2, peak n). No reducing sugars (glucose, fructose, galactose) were accumulated in the buckwheat embryo during seed development and maturation.

Evidence for the identity of the fagopyritol peaks is presented in FIGS. 3A-C. Before hydrolysis, the fagopyritols are readily apparent (FIG. 3A, peaks e, f, k, 1). After hydrolysis with α-galactosidase, an enzyme that hydrolyses galactosyl bonds in the (alpha)-position, peaks e to 1 are gone (FIG. 3B) whereas D-chiro-inositol (FIG. 3B, peak a) increases greatly, and myo-inositol (peak b) increases slightly, but no other free cyclitols appear. Sucrose (FIG. 3B, peak d) does not change significantly, but anomeric, forms of galactose (FIG. 3B, peaks marked “1”) appear. Little sucrose and galactose result from hydrolysis of raffinose and stachyose, because these sugars are present only in trace amounts. Therefore, nearly all of the galactose was released from galactosyl cyclitols. myo-inositol increases slightly (FIG. 3B, peak b) due to hydrolysis of galactinol (FIG. 3a, peak g) and traces of digalactosyl myo-inositol (FIG. 2, peak m). The fagopyritols were resistant to β-galactosidase (EC 3.2.1.23) for 24 h at 37° C. and no galactose was released. This pattern confirms that peaks e, f, k, and 1 in FIG. 3A contained α-galactosyl linkages only. Peak 1 (Fagopyritol B2) had a retention time identical to O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol (Shiomi et al., “A New Digalactosyl Cyclitol from Seed Balls of Sugar Beet,” Agric. Biol. Chem., 52:1587-1588 (1988), which is hereby incorporated by reference) extracted from embryos of mature sugarbeet (Beta vulgaris L.) seed. After hydrolysis with a strong acid (2 N TFA), sucrose also is hydrolyzed resulting in anomeric forms of glucose (FIG. 3C, two peaks marked “2”) and fructose (FIG. 3C, three peaks marked “3”). The internal standard (peak c) was added after hydrolysis. After correction for galactinol and the assumption that Fagopyritol A2 and Fagopyritol B2 are digalactosyl chiro-inositols, the products from acid hydrolysis of the tissue extract have a calculated ratio of 1.00 D-chiro-inositol to 0.93 galactose residues for Fagopyritol A1 plus Fagopyritol B1 (peaks e and f) providing evidence that both are galactosyl chiro-inositols.

For additional confirmation, fagopyritols were separated by liquid column chromatography (FIG. 4). α-Galactosidase hydrolysis products of Fagopyritol B1 (FIG. 4, peak d), after correction for galactose recovery, gave a ratio 1.00 D-chiro-inositol to 1.08 galactose confirming that Fagopyritol B1 is an α-galactosyl D-chiro-inositol. Gas chromatography analysis of the TMS-product of peak f in FIG. 4 had one peak corresponding to Fagopyritol B2. After partial hydrolysis with 2 N TFA, peaks corresponding to Fagopyritol B2, Fagopyritol B1, galactosyl galactose, D-chiro-inositol, and galactose were observed. After correction for galactose recovery, a ratio of 1.00 D-chiro-inositol to 1.93 galactose residues confirms that Fagopyritol B2 is a digalactosyl D-chiro-inositol. α-Linkage of galactose in the fagopyritols was verified also by ¹H-NMR. The ratio of D-chiro-inositol to galactose and the position of the peaks suggest that peaks e and f are probably different position isomers of galactosyl D-chiro-inositol, and peaks k and 1 are the corresponding digalactosyl D-chiro-inositols. GC-MS analysis demonstrates that Fagopyritol A1 and Fagopyritol B1 are closely related but have distinct fragmentation patterns. Structures for the galactinol series are depicted in FIG. 5.

The presence of D-chiro-inositol in Fagopyritol B1 and Fagopyritol A1 was carried out using high resolution gas chromatography and a chiral capillary column. Authenic D-chiro-inositol and L-chiro-inositol standards and the acid hydrolysis products (2 N TFA, 70° C. for 3 h) of pure Fagopyritol B1 and pure Fagopyritol A1 were derivatized with pentafluoropropionic anhydride (“PFPN”):acetonitrile (1:1, v/v). Derivatized products were co-chromatographed with standards of authentic D-chiro-inositol and L-chiro-inositol using high resolution gas chromatography with a chiral capillary column (Chirasil-Val, Alltech, Deerfield, Ill.; 25 m length, 0.25 mm inside diameter, 0.16 gin film thickness) run at 100° C. isothermally. Injection port temperature was 130° C. and flame ionization detector (“FID”) temperature was 130° C. D-chiro-inositol in Fagopyritol B1 and Fagopyritol A1 co-chromatographed with authentic D-chiro-inositol (4.6 minutes retention time) but not with authentic L-chiro-inositol (4.9 minutes retention time). Fagopyritol B1 and Fagopyritol A1 were confirmed to have only D-chiro-inositol with a retention time of 4.6 minutes.

In addition, the exact structure of the galactosyl ring, the D-chiro-inositol ring, and the (α-linkage from the anomeric carbon of galactose to carbon-2 on the D-chiro-inositol ring was confirmed by NMR experiments with pure Fagopyritol B1. See Szczencinski et al., “NMR Investigation of the Structure of Fagopyritol B1 From Buckwheat Seeds,” Bulletin of the Polish Academy of Sciences Chemistry, 40:9-13 (1998), which is hereby incorporated by reference.

Example 3 Accumulation of Sugars and Cyclitols During Seed Development and Maturation

Buckwheat embryos accumulate maximum water content at 12 DAP whereas maximum fresh weight occurs at 16 DAP, and no significant increase in embryo dry matter occurs after 16 DAP (Table 1). The slight decrease in embryo dry matter noted at the latter stages of seed maturity probably reflect the difficulty encountered in physically removing all remnants of the cotyledons, which are entwined throughout the endosperm (as illustrated in Marshall et al., “Buckwheat: Description, Breeding, Production, and Utilization,” Adv. Cereal Sci. Tech., 5:157-210 (1982), which is hereby incorporated by reference) and adhere to the seed coat in the desiccated seed.

TABLE 1 Fresh weight Dry weight DAP (Mg) (Mg) Water (Mg) Water (g/gFW)  6  0.43 ± 0.00 0.11 0.32  .750  8  1.60 ± 0.29 0.48 1.12 0.697 10  7.68 ± 0.77 2.74 ± 0.77 4.94 ± 0.27 0.658 = L-0.042 12 10.36 ± 1.13 4.38 ± 0.63 5.M ± 0.55 0.581 = L-0.019 16 11.67 ± 0.44 6.10 ± 0.50 5.57 ± 0.37 0.479 = L-0.033 20 10.18 ± 0.52 6.42 ± 0.28 3.76 ± 0.33 0.368 ± 0.018 24  7.27 ± 0.99 5.67 ± 0.44 1.60 ± 0.65 0.206 ± 0.056 28  5.83 ± 0.50 5.07 ± 0.67 0.77 ± 0.23 0.137 ± 0.045 32  5.43 ± 0.28 5.13 ± 0.24 0.30 ± 0.06 0.055 ± 0.008

Table 2 shows the amounts of various carbohydrates and cyclitols present in buckwheat embryos (expressed in units of μg/embryo) at various stages during seed development and maturation. Fructose and glucose were present only in very young embryos, at 6 and 8 DAP, but not at later stages of embryo development and maturation (Table 2). D-chiro-inositol and myo-inositol were present from the earliest stages throughout embryo development and maturation (Table 2) whereas pinitol or other O-methyl cyclitols were not detected at any stage of embryo development and maturation. Sucrose increased dramatically between 8 and 10 DAP (Table 2), corresponding to the beginning of very rapid growth of the embryo (Table 1), and continued to increase at a slower rate through 16 DAP (Table 2). Fagopyritol B1 and its isomeric form, Fagopyritol A1, were first detected at 12 DAP and accumulated very rapidly between 12 and 16 DAP (Table 2). Fagopyritol A2 and Fagopyritol B2 were first detected at 16 DAP and accumulated rapidly to 20 DAP. Fagopyritol A3 accumulated at 20 DAP (Table 2) during the early phase of embryo desiccation (50 to 35% water concentration; Table 1). Total soluble sugars and cyclitols accumulated in two waves: first, 8 to 10 DAP in association with rapid embryo growth and sucrose accumulation and, second, 12 to 16 DAP in association with a rapid accumulation of Fagopyritol B1 (Table 2).

TABLE 2 Component 6 DAP 8 DAP 10 DAP 12 DAP 16 DAP fructose 1.90 ± 0.34 1.12 ± 0.02 0 0 0 glucose 2.44 ± 0.53 3.90 ± 0.02 0 0 0 D-chiro-inositol 1.07 ± 0.17 3.60 ± 0.58 3.85 ± 1.42 1.68 ± 0.75 9.64 ± 0.68 myo-inositol 0.03 ± 0.01 0.12 ± 0.49 1.06 ± 0.14 1.75 ± 0.13 3.42 ± 0.56 sucrose 6.79 ± 0.43 15.00 ± 0.49  212.2 ± 34.8  262.0 ± 46.9  295.9 ± 38.5  Fagopyritol A1^(a) 0 0 0 0.95 ± 0.95 35.0 ± 4.24 Fagopyritol B1^(a) 0 0 0 4.34 ± 2.45 162.2 ± 49.6  galactinol 0 0 0 0 7.84 ± 1.37 raffinose 0 0 0 0 0 Fagopyritol A2^(b) 0 0 0 0 7.86 ± 3.96 Fagopyritol B2^(b) 0 0 0 0 6.51 ± 3.51 digalactosylmyo-inositol^(b) 0 0 0 0 0.43 ± 0.43 stachyose 0 0 0 0 0 Fagopyritol A3^(c) 0 0 0 0 0 Total 12.23 23.74 217.1 ± 33.5  266.4 ± 48.2  528.8 ± 42.2  suc:Fagopyritol B1^(d) (ratio) ∞ ∞ ∞ 60.36 1.82 suc:non-suc^(d) (ratio) 1.25 1.72 43.31 59.54 1.27 Component 20 DAP 24 DAP 28 DAP 32 DAP fructose 0 0 0 0 glucose 0 0 0 0 D-chiro-inositol 3.77 ± 0.34 3.49 ± 0.51 2.74 ± 0.95 2.26 ± 0.38 myo-inositol 2.51 ± 0.14 2.71 ± 0.30 2.38 ± 0.12 3.08 ± 0.20 sucrose 319.8 ± 19.8  262.0 ± 46.9  272.3 ± 18.1  300.0 ± 9.71  Fagopyritol A1^(a) 32.88 ± 4.24  28.38 ± 3.07  21.38 ± 4.24  24.38 ± 4.43  Fagopyritol B1^(a) 194.2:20.3 163.5 ± 9.1  123.2 ± 22.4  158.2 ± 30.0  galactinol 4.65 ± 0.51 4.26 ± 2.33 5.73 ± 0.99 4.18 ± 0.09 raffinose 0.34 ± 0.29 0.84 ± 0.09 0.26 ± 0.14 1.10 ± 0.45 Fagopyritol A2^(b) 17.36:2.01 12.71:1.37 8.74:1.06 12.67 ± 2.76  Fagopyritol B2^(b) 18.56 ± 2.67  12.63 ± 1.71  10.36 ± 0.74  15.52 ± 3.33  digalactosylmyo-inositol^(b) 1.22 ± 0.83  0.69:0.44 0.99 ± 0.53 1.43 ± 0.88 stachyose 0 0 0 0 Fagopyritol A3^(c) 4.79 ± 0.56 3.19 ± 0.39 3.13 ± 0.43 3.43 ± 1.71 Total 600.3 ± 9.5  495.0 ± 25.1  451.1 ± 11.4  426.2 ± 37.7  suc:Fagopyritol B1^(d) (ratio) 1.65 1.60 2.21 1.89 suc:non-suc^(d) (ratio) 1.14 0.88 1.52 1.32 ^(a)Calculated on the galactinol standard. ^(b)Calculated on the raffinose standard. ^(c)Calculated on the staehyose standard. ^(d)Ratio of sucrose to Fagopyritol B1 and ratio of sucrose to non-sucrose was calculated on mean values.

Example 4 Distribution of Sugars and Cyclitols in Buckwheat Seeds

The axis was found to be 8% of the embryo fresh weight, whereas the cotyledons were 92% of the embryo fresh weight at 20 DAP (Table 3). The concentration of all components were higher in the axis than in the cotyledons (Table 3). Sucrose, cyclitols, and their derivatives accumulated to 15% of the dry weight of the axis of buck-wheat embryo compared to 8% of the cotyledon dry weight (Table 3). Fagopyritol B1 concentration in axis tissues was double that in the cotyledons, and the ratio of sucrose to Fagopyritol B1 was 1.07 (w:w) in the axis compared to 1.64 in the cotyledons. The flatulence-producing oligosaccharides, raffinose and stachyose, were present in the axis tissues but only in trace amounts in the cotyledons (Table 3). Galactinol is the galactose donor to sucrose (DP-2) to form raffinose (galactosyl sucrose, a DP-3) and to raffinose to form stachyose (galactosyl sucrose, a DP-4).

TABLE 3 Axis Cotyledons Carbohydrate or cyclitol (μg/embryo) (mg/g FW) (mg/g DW) (μg/embryo) (mg/g FW) (mg/g DW) D-chiro-inositol 0.76 ± 0.12 0.89 ± 0.14 1.41 ± 0.02 3.26 ± 1.08 0.32 ± 0.11 0.51 ± 0.17 myo-inositol 0.36 ± 0.03 0.42 ± 0.06 0.66 ± 0.09 3.15 ± 0.14 0.31 ± 0.02 0.49 ± 0.03 Sucrose 34.77 ± 3.25  40.35 ± 12.50 63.89 ± 13.96 275.4 ± 73.7  26.59 ± 6.24  42.11 ± 9.88  Fagopyritol A1^(a) 5.21 ± 0.12 6.10 ± 0.44 9.66 ± 0.69 32.26 ± 4.99  3.20 ± 0.60 5.06 ± 0.95 Fagopyritol B1^(a) 32.47 ± 1.67  38.16 ± 3.91  60.43 ± 6.19  167.6 ± 34.2  16.70 ± 3.95  26.44 ± 6.25  Galactinol 0.87 ± 0.14 1.02 ± 0.19 1.62 ± 0.30 5.49 ± 1.56 0.54 ± 0.14 0.85 ± 0.22 Raffinose 0.14 ± 0.02 0.16 ± 0.02 0.25 ± 0.03 0 0 0 Unknown^(b) 0.55 ± 0.05 0.65 ± 0.10 1.03 ± 0.16 0.79 ± 0.46 0.14 ± 0.03 0.22 ± 0.05 Fagopyritol A2^(b) 1.66 ± 0.01 1.95 ± 0.09 3.08 ± 0.14 17.40 ± 1.04  1.72 ± 0.16 2.72 ± 0.25 Fagopyritol B2^(b) 2.87 ± 0.19 3.38 ± 0.41 5.35 ± 0.64 15.94 ± 0.79  1.57 ± 0.13 2.48 ± 0.20 Digalactosyl 0.33 ± 0.03 0.38 ± 0.03 0.60 ± 0.05 1.66 ± 0.75 0.06 ± 0.03 0.09 ± 0.05 myo-linositol^(b) Stachyose 0.89 ± 0.07 1.05 ± 0.13 1.66 ± 0.20 tr tr tr Fagopyritol A3^(c) 1.21 ± 0.03 1.41 ± 0.11 2.23 ± 0.17 3.98 ± 0.51 0.39 ± 0.01 0.61 ± 0.02 Total 82.08 ± 2.24  95.92 ± 4.92  151.89 ± 7.79  527.4 ± 39.8  51.36 ± 2.75  81.33 ± 4.35  (ratio) (ratio) suc:Fagopyritol B1^(d) 1.07 1.64 suc:non-suc^(d) 0.73 1.09 (mg/axis) (mg/cots) fresh weight 0.86 ± 0.04 10.20 ± 0.35  ^(a)Calculated on the galactinol standard. ^(b)Calculated on the raffinose standard. Unknown has retention time ratio of 2.44 relative to phenyl 1-D-glucoside. ^(c)Calculated on the stachyose standard. ^(d)Ratio of sucrose to Fagopyritol B1 and ratio of sucrose to non-sucrose was calculated on mean values.

The endosperm fraction, including the aleurone layer and bran fraction of maternal seed coat-nucellus (spermoderm) tissues, was 74% of the dehulled groat, and the embryo was 26% of the dehulled groat (Table 4). The embryo fraction contained 71.4% of the total soluble carbohydrates and cyclitols, and the endosperm fraction contained 28.6%. The embryo contained five times as much Fagopyritol B1 (403 μg) as the endospern fraction (76 μg) (Table 4). Most of the sugars, cyclitols, and cyclitol derivatives in the endosperm fraction were probably in the aleurone layer. Some could have been in fragments of cotyledons that could not be separated completely from the endosperm fraction of mature dry groats. The concentration of soluble components in the endosperm fraction was low because starch is the major storage component.

TABLE 4 Carbohydrate Endosperm Embryo or cyclitol (mg/g dry weight) (mg/g dry weight) D-chiro-inositol 0.25 ± 0.03 0.80 ± 0.16 myo-inositol 0.10 ± 0.01 0.27 + 0.02 sucrose 6.99 ± 0.37 30.5 ± 0.84 Fagopyritol A1^(a) 0.41 ± 0.04 2.10 ± 0.27 Fagopyritol B1^(a) 2.77 ± 0.37 41.16 ± 2.97  galactinol 0.04 ± 0.03 0.29 ± 0.03 Raffinose 0 tr Fagopyritol A2^(b) 0.18 ± 0.03 0.86 ± 0.05 Fagopyritol B2^(b) 0.34 ± 0.03 1.50 ± 0.20 Digalactosyl myo-inositol^(b) 0.05 ± 0.02 0.13 ± 0.03 Stachyose 0 tr Fagopyritol A3^(c) tr 0.21 ± 0.07 TOTAL 11.19 ± 0.79  77.82 ± 4.20  (mg) dry weight 27.3 ± 1.0 9.8 ± 0.8 ^(a)Calculated on the galactinol standard. ^(b)Calculated on the raffinose standard. ^(c)Calculated on the stachyose standard.

Example 5 Cultivar and Species Differences in Cyclitols and Soluble Carbohydrates

The concentration of total soluble carbohydrates and cyclitols in mature dehulled groats was about the same in ‘Mancan’ and ‘Manor’ common buckwheat (Table 5) and in ‘Tartary’ buckwheat. The concentration of sucrose was the same in groats of all three genotypes. Fagopyritol B1 was highest in concentration in ‘Mancan’, lower in ‘Manor’, and only 20% as high in ‘Tartary’ as in ‘Mancan’. ‘Tartary’ had two unknown compounds not found in ‘Mancan’ or ‘Manor’, whereas ‘Manor’ had higher concentrations of Fagopyritol A2, Fagopyritol B2, and Fagopyritol A3 (Table 5). Part of the differences may have been related to the environment of production, since the three genotypes were not grown under the same environmental conditions. However, the high concentration of unknown 1 and the low concentration of Fagopyritol B1 in groats of ‘Tartary’ buckwheat were probably a reflection of genetic differences. The shift to higher concentrations of the higher galactosyl oligomers in groats of ‘Manor’ buckwheat may have reflected environmental influences.

TABLE 5 Retention Carbohydrate time Mancan Manor or cyclitol ratio (Rt) (mg/g dry weight) (mg/g dry weight) D-chiro-inositol 0.51 0.40 ± 0.07 0.26 ± 0.06 myo-inositol 0.72 0.10 ± 0.01 0.14 ± 0.01 Sucrose 1.71 13.17 ± 0.34  12.57 ± 1.21  unknown^(a) 1.76 0 0 unknown 2^(a) 1.81 0 0 Fagopyritol A1^(a) 1.96 0.86 ± 0.11 1.10 ± 0.10 Fagopyritol B1^(a) 2.01 12.86 ± 1.08  8.11 ± 0.32 galactinol 2.05 0.10 ± 0.01 0.15 ± 0.03 raffinose 2.35 tr tr Fagopyritol A2^(b) 2.59 0.35 ± 0.02 0.82 ± 0.16 Fagopyritol B2^(b) 2.63 0.64 ± 0.06 1.43 ± 0.23 digalactosyl myo- 2.67 0.07 ± 0.02 0.21 ± 0.08 inositol^(b) stachyose 2.86 tr tr Fagopyritol A3^(c) 3.16 0.06 ± 0.02 3.38 ± 0.08 TOTAL 28.58 ± 1.64  25.58 ± 1.48  (mg/seed) (mg/seed) dry weight 37 ± 1  23 ± 1  ^(a)Calculated on the galactinol standard. ^(b)Calculated on the raffinose standard. ^(c)Calculated on the stachyose standard.

Sensitivity to the flavanols, quercetin, and rutin, was quite low by the assay methods used herein. Rutin, commonly present in buckwheat leaves, was detected only in trace amounts in groats from mature seeds of common buckwheat, either ‘Mancan’ or ‘Manor’ cultivars. In contrast, groats from mature seeds of ‘Tartary’ buckwheat contained about 7.14 mg of rutin per gram of dry weight, calculated with a rutin standard and, after sample hydrolysis, with quercetin and rhamnose standards. Products from acid hydrolysis of rutin were quercetin, rhamnose, and glucose. Quercetin and rhamnose were detected after acid hydrolysis (2 N TFA for 3 h at 70° C. of extracts from ‘Tartary’ buck-wheat groats, but neither was found in acid hydrolysis products of extracts from embryos of common buckwheat. Glucose was an acid hydrolysis product of sucrose also and, therefore, could not be used for assay of rutin in tissue extracts. Because the embryo could not be separated from the endosperm of mature dry ‘Tartary’ buckwheat seeds, 5 embryo tissues could not be analyzed.

Example 6 Total Fagopyritols, Free D-chiro-inositol, and Total D-chiro-inositol in Buckwheat Groats, Achenes and Milling Fractions

Among the milling fractions, the “purified bran” fraction had a high concentration of embryo fragments that adhered to the seed coats. In the absence of hulls, this fraction had the highest concentration of fagopyritols and total D-chiro-inositol among the milling fractions (Table 6). Total fagopyitols and total D-chiro-inositol concentrations in the “purified bran” were double those of the next highest milling fraction (bran #1). The concentrations of total fagopyritols and total D-chiro-inositol declined in milling fractions in the order of decreasing concentration of embryo tissues in that milling fraction (Table 6). This descending order was “purified bran”>“bran #1” (with hulls)>“bran #2” (with fewer hulls)>whole groat flour>“fancy flour” (much starch)>“cracks” (with few embryo fragments). The “cracks” fraction was very difficult to pulverize and extract. The starchy endosperm of the “cracks” fraction resembled partially germinated seeds that had been dried. Upon wetting of dry seeds, α-amylase was released by cotyledons into the starchy endospern and began to digest the starch. This sticky starch became very hard upon drying. It was postulated that some of the “cracks” come from seeds that re-wetted on the plant before harvest. The fagopyritols disappeared within 12 to 18 hours of re-wetting, and starch degradation had already started by that time. By analogy, the cracks fraction had few embryo fragments, and those fragments that were present may have reduced fagopyritols if the seed was re-wetted before harvest.

TABLE 6 Minn-Dak Minn-Dak Minn-Dak Purified Minn-Dak Minn-Dak fancy flour Minn-Dak cracks whole groat flour Soluble Carbohydrate bran (mg/100 g) bran #1 (mg/100 g) bran #2 (mg/100 g) (mg/100 g) (mg/100 g) (mg/100 g) D-chiro-inositol  72.6 ± 13.2 57.8 ± 0.8 53.5 ± 2.8 14.9 ± 0.4 6.0 ± 0.4 23.3 ± 0.5 myo-inositol 35.8 ± 1.4 23.7 ± 1.0 21.5 ± 1.0  5.3 ± 0.8 2.9 ± 0.3 14.0 ± 0.5 Sucrose 2177.7 ± 128.7 1282.0 ± 61.7  1045.3 ± 42.5  344.3 ± 30.9 190.5 ± 7.2  855.6 ± 30.4 Fagopyritol A1 208.5 ± 10.7 92.9 ± 4.9 78.2 ± 3.8 30.3 ± 2.9 10.8 ± 0.5  49.7 ± 3.7 Fagopyritol B1 1335.51-68.0 600.3 ± 37.1 510.7 ± 24.4 180.1 ± 10.3 68.5 ± 4.1  346.2 ± 24.2 Galactinol 32.2 ± 1.7 25.4 ± 4.0 19.6 ± 1.5  3.8 ± 0.4 3.1 ± 0.3 18.2 ± 1.3 Fagopyritol A2 89.7 ± 3.6 57.2 ± 3.0 51.7 ± 7.6 14.6 ± 0.9 8.0 ± 0.7 32.0 ± 2.6 Fagopyritol B2 120.5 ± 2.8  76.0 ± 1.7 73.6 ± 9.0 20.3 ± 2.6 14.5 ± 1.1  47.0 ± 4.1 Digalactosyl 18.9 ± 0.5 23.4 ± 0.8 20.0 ± 2.7  4.7 ± 0.8 6.1 ± 1.0 11.6 ± 0.9 Fagopyritol A3 58.2 ± 0.8 39.5 ± 5.8 40.3 ± 5.4 10.4 ± 0.6 9.6 ± 1.7 23.2 ± 2.9 Total soluble carbohydrates 4149.5 ± 211.1 2278.2 ± 111.5 1914.4 ± 94.0  628.8 ± 43.9 319.9 ± 15.6  1420.7 ± 63.2  Total Fagopyritols 1812.3 ± 81.5  865.9 ± 51.7 754.5 ± 46.1 255.7 ± 14.3 111.4 ± 7.3  498.0 ± 34.7 Total D-chiro-inositol 929.2 ± 43.2 458.7 ± 23.2 399.8 ± 20.5 134.4 ± 7.2  55.5 ± 3.4  253.4 ± 15.4 Free D-chiro-inositol 72.6 ± 3.2 57.8 ± 0.8 53.5 ± 2.8 14.9 ± 0.4 6.0 ± 0.4 23.3 ± 0.5 Water (g per 100 g of fresh wt.) 11.6 ± 03  10.0 ± 0.2  9.0 ± 0.2 12.3 ± 0.2 11.4 ± 0.2  11.8 ± 0.3

Example 7 Temperature Effects on Accumulation of Sugars and Cyclitols

The sugar composition of embryos (cotyledons and axis together) is reported for embryos removed from mature seeds harvested from plants grown at 18 and 25° C. The major soluble carbohydrates in buckwheat embryos were sucrose and Fagopyritol B1, accounting for 85% of the total at 18° C. (Table 7). Since embryo dry weight was similar between the two temperatures, the endosperm accumulated an average of 15 mg more dry weight per seed at 18° C. than at 25° C. The temperature effect on the embryo soluble carbohydrates was quite interesting. Embryos of seeds grown at 18° C. had reduced sucrose, 2.5 times as much Fagopyritol B1, and three times as much stachyose as those grown at 25° C. Fagopyritol A2 and Fagopyritol B2 accumulated to higher levels at 25° C. (Table 7). When grown in the greenhouse where temperatures often exceed 25° C., Fagopyritol A3 was detectable at 20 DAP (Table 2). This pattern suggests that low temperature favors accumulation of Fagopyritol B1 while higher temperature favors accumulation of the higher galactosyl oligomers of fagopyritol. The reverse pattern occurred for stachyose. Stachyose accumulation was favored by cool temperature (Table 7) and was not detected in embryos from seeds maturing at the warmer temperatures in the greenhouse (Table 2).

TABLE 7 Carbohydrate seeds grown at 18° C. seeds grown at 25° C.^(a) or cyclitol (μg/g embryo) (μg/g embryo) D-chiro-inositol 13.45 ± 0.46  1.69 ± 0.10 myo-inositol 1.60 ± 0.32 1.84 ± 0.10 sucrose 211.60 ± 14.0  252.70 ± 6.40  Fagopyritol A1^(b) 34.86 ± 5.89  15.65 ± 0.98  Fagopyritol B1^(b) 263.50 ± 9.01  102.60 ± 2.95  galactinol 0 0.87 ± 0.44 raffinose 1.98 ± 0.49 2.27 ± 0.92 unknown^(c) 1.45 ± 0.35 2.94 ± 0.66 Fagopyritol A2^(c) 5.19 ± 1.68 10.81 ± 1.11  Fagopyritol B2^(c) 6.17 ± 1.12 15.86 ± 2.33  digalactosyl myo-inositol tr tr stachyose 17.30 ± 7.45  5.64 ± 1.69 Fagopyritol A3 tr tr TOTAL 557.10 ± 36.2  412.90 ± 9.05  (mg/embryo ± SB) (mg/embryo ± SE) embryo dry weight 5.42 ± 0.17 5.75 ± 0.52 (mg/g dry weight) (mg/g dry weight) TOTAL soluble 0.103 (10.3%) 0.072 (7.2%) (ratio) (ratio) sucrose/Fagopyritol B1 0.80 2.46 sucrose/non-sucrose 0.61 1.58 ^(a)Mature dry seeds were 473 mg (18° C.) and 32 ± 3 mg (25° C.) before embryo excision; three replicates. ^(b)Calculated on the galactinol standard. ^(c)Calculated on the raffinose standard. Unknown has retention time ratio of 2.44 relative to phenyl a-D-glucoside.

Example 8 Relationship of Fagopyritol B1 to Desiccation Tolerance During Embryo Development

Developing embryos in buckwheat seeds became desiccation tolerant at 12 DAP (FIG. 6). Freshly harvested seeds exhibit a high degree of dormancy (Samimy et at., “Seed Dormancy in Common Buckwheat (Fagopyruni esculentum Moench,” Plant Varieties and Seeds, 7:17-22 (1994), which is hereby incorporated by reference). Seeds not germinated after 16 d on wet paper towels at 25° C. were considered dormant and contained 76 to 87% as much Fagopyritol B1 (Table 8) as in planta seeds at 20 and 24 DAP (Table 2), suggesting that dormancy slows the mobilization of Fagopyritol B1. The development of desiccation tolerance at 12 DAP (FIG. 6) occurred in association with the accumulation of Fagopyritol B1 at 12 to 16 DAP (Table 2).

TABLE 8 20 DAP dormant^(a) 24 DAP dormant^(a) Carbohydrate or cyclitol (μg/embryo) (μg/embryo) D-chiro-inositol 8.05 ± 1.03 1.94 ± 0.68 myo-inositol 2.12 ± 0.30 1.94 ± 0.38 sucrose 224.1 ± 49.3  144.8 ± 24.6  Fagopyritol A1^(b) 23.36 ± 0.91  25.47 ± 2.80  Fagopyritol B1^(b) 148.1 ± 17.6  143.4 ± 19.7  galactinol 1.30 ± 0.65 1.51 ± 0.75 raffinose 0 0 unknown, Rt = 2.49^(c) 1.19 ± 0.60 0.76 ± 0.76 unknown, Rt = 2.51^(c) 4.05 ± 0.72 1.92 ± 0.96 Fagopyritol A2^(c) 7.28 ± 1.36 8.65 ± 0.71 Fagopyritol B2^(c) 7.37 ± 1.11 8.33 ± 1.17 digalactosyl myo-inositol tr 0.11 ± 0.11 Stachyose 0 0 Fagopyritol A3 0 0 TOTAL 427.0 ± 43.0  338.8 ± 3.7  ^(a)Fresh seeds were harvested at 20 or 24 DAR Seeds not germinating after 16 days on wet paper towels at 25° C. are considered to be dormant. ^(b)Calculated on the galactinol standard. ^(c)Calculated on the raffinose standard. Unknowns have retention time ratios of 2.49 and 2.51 relative to phenyl a-D-glucoside.

When immature buckwheat seeds were harvested at 10 DAP, desiccation tolerance was induced by slow drying under controlled relative humidities (FIG. 7A). Acquisition of desiccation tolerance during slow drying of immature seeds (precocious maturation or induced maturation) was closely associated with the accumulation of Fagopyritol B1 during the first day of slow drying (FIG. 7C). D-chiro-inositol declined rapidly during the first day of slow drying (FIG. 7B). myo-inositol remained constant (FIG. 7B), but galactinol increased during the second day (FIG. 7C). Small amounts of Fagopyritol A2, Fagopyritol B2, and digalactosyl myo-inositol accumulated to the fifth day (FIG. 7D). Sucrose declined during the first day and then remained constant (FIG. 7E).

Similarly, when held at high relative humidity, immature 1o-DAP dehulled buckwheat seeds became desiccation tolerant after 1 day (FIG. 8A) in association with the accumulation of Fagopyritol B1 (FIG. 8C) and the decline in D-chiro-inositol (FIG. 8B). Galactinol increased during the second day at high relative humidity (FIG. 8C). Small amounts of Fagopyritol A2 and Fagopyritol B2 accumulated 2-5 d, and digalactosyl myo-inositol accumulated 2-6 d (FIG. 8D). Fagopyritol A3 was detected only after 6 days. Sucrose increased slowly after the first day (FIG. 8E). This pattern was in contrast to axes of soybean seeds, which do not become desiccation tolerant during incubation at high relative humidity and do not accumulate stachyose during incubation at high relative humidity (Blackman I, which is hereby incorporated by reference). It appears that Fagopyritol B1 can accumulate under very mild conditions of desiccation, but accumulation of the raffinose series of oligosaccharides may require more desiccation.

Example 9 Relationship of Fagopyritol B1 to Loss of Desiccation Tolerance During Germination

The relationship of loss of Fagopyritol B1 to loss of desiccation tolerance (rapid drying at 12% relative humidity and 22° C. was determined in axis and cotyledon tissues during germination of dehulled mature buckwheat seeds (‘Mancan’). Within 6 h after the start of imbibition, Fagopyritol B1 started to disappear from axis tissues. By 12 h, only low levels remained, and by 18 h of germination, all fagopyritols completely disappeared except for trace amounts of higher oligomers (FIG. 9E). The disappearance of Fagopyritol B1 was closely associated with the onset of rapid germination and the loss of desiccation tolerance as measured by “normal” growth of the hypocotyl-radicle axis (primary root growth) upon rehydration (FIG. 9A). Sucrose levels declined and then increased after 48 h, reflecting mobilization of sugars from the cotyledons (FIG. 9C). The sucrose:Fagopyritol B1 ratio increased very rapidly after 12 h in association with the loss of desiccation tolerance and the onset of germination. In contrast to sucrose, D-chiro-inositol increases after 12 h, reflecting the hydrolysis of the fagopyritols (FIG. 9C). Galactose was not detected during mobilization of the fagopyritols but apparently was converted rapidly to other products. Fructose and glucose were present in axis tissues after 18 h (FIG. 9I) when axis tissues were growing rapidly (FIG. 9B). Fructose and glucose were the only reducing sugars in axis tissues (FIG. 9I).

During germination maltose, maltotriose, and maltotetraose (FIG. 9J) were present in cotyledon tissues in addition to increasing levels of sucrose (FIG. 9H) reflecting the degradation of starch. D-chiro-inositol increased five fold between 6 and 18 h of germination (FIG. 9B), reflecting the degradation of fagopyritols in the cotyledons (FIG. 9B). The total amount of D-chiro-inositol (free and bound) in cotyledons declined during germination, suggesting the occurrence of transport to axis tissues or metabolism into other products. The high amount of sucrose in the cotyledons at 18 to 48 h of germination reflected the mobilization of starch in the endosperm and the absorption of hydrolysis products by the cotyledons (FIG. 9H—J). Cotyledon tissues were imbedded in the endosperm and surrounded the starchy endospern during germination. The lower level of Fagopyritol B1 and total soluble carbohydrates in the cotyledons at 0 h (FIG. 9F) indicated that part of the outer cotyledon, in contact with the testa (seed coat) of dry seeds, was lost during dissection of the embryo tissues from mature dry seeds.

Example 10 Materials For Fractionation Experiments

Samples of 100 dehulled seeds (about 2.5 g) were pulverized and extracted with ethanol:water (50:50). Extracts were centrifuged repeatedly, concentrated to a few ml in volume, and passed through a 10,000 M_(r) cut-off filter. Ethanol was added to concentrated samples and placed in a freezer to facilitate precipitation of fagopyritols with the aim of concentrating Fagopyritol A3 from the crude extracts. In most cases, Fagopyritol A3 was lost or was below the level of detection in samples from the preparations.

About 97% of the sucrose was removed by invertase. Glucose, fructose, and galactose increased, and raffinose and stachyose were still present, probably because the invertase enzyme preparation was not sufficiently pure. Attempts to destroy glucose and fructose by different methods were only partially successful.

Example 11 Fractionation on P2 Gels

Several sets of extracts were pooled, concentrated, and clarified by centrifugation and ultrafiltration. The combined extracts of 1081 seeds was concentrated to 3 ml. The concentrated preparation of soluble carbohydrates was loaded on a Bio-Gel P2 gel column (3 m×40 cm) and eluted with degassed water at 65° C. Early fractions were brown, and subsequent fractions were yellow in coloration. Most of the sugars and fagopyritols eluted in three 5-ml fractions and were still contaminated with brown colored material. These fractions were pooled, concentrated, and loaded onto a second Bio-Gel P2 gel column at 20° C. Fractions enriched in Fagopyritol A3 were pooled; those enriched in Fagopyritols A1 and B1 were pooled; and those enriched in Fagopyritols A1, B1, A2, and B2 and sucrose were pooled. The flow rate of the column decreased as the quantity of brown colored materials (probably polyphenolic materials) increased.

Extracts from 1300 seeds (about 35 g) were clarified by centrifugation, concentrated, and passed through a 10,000 M_(r) cut-off filter. After concentration to 1.5 ml, the sample was ultrafiltered, and the concentrated extracts were chromatographed on P2 columns. Fractions enriched in Fagopyritol A3 and fractions enriched in other fagopyritols were combined from each of the extracts. Boiling before centrifugation resulted in swelling of starch, loss of extractable volume, and increased coloration in extracts. None of the columns resulted in efficient separation of the different fagopyritols.

Example 12 Fractionation on 3-Aminopropvl-functionalized Silica Gel Columns

A 0.9 cm×30 cm column of 3-aminopropyl-functionalized (9% functionalized) silica gel (Sigma-Aldrich) was used for fractionation of fagopyritols in subsequent experiments. Enriched fractions from the P2 gel columns were loaded on the first five amino columns.

For column 3, a sample enriched in Fagopyritol A3 after partial purification on a P2 gel column was evaporated to dryness, dissolved in 2 ml of water, and passed through a PVP column. The eluate was evaporated to dryness, dissolved in 7 ml of acetonitrile:water (70:30), and loaded onto a 0.9 cm×30 cm 3-aminopropyl-functionalized silica gel column. Stepwise elution was with 350 ml of acetonitrile:water (70:30), 150 ml of acetonitrile:water (60:40), and 50 ml of acetonitrile:water (50:50). The column was regenerated with 30 ml of acetonitrile:water (60:40) and 30 ml of acetonitrile:water (75:25). Fractions (5 ml) were collected and 0.5 ml samples from the collected fractions were evaporated to dryness and derivatized with TMSI:pyridine (1:1). The TMS-carbohydrates were analyzed by high resolution gas chromatography. Fagopyritol A3 was clearly separated from the other fagopyritols (FIG. 10). The pooled fractions containing “pure” Fagopyritol A3 were evaporated to dryness and stored for rechromatography to remove traces of contaminants. Fractions of “pure” Fagopyritol A2 or “pure” Fagopyritol B2 were pooled. Fractions of mixtures of Fagopyritols A2 and B2 were also pooled for recycling.

For column 4, a sample containing a mixture of fagopyritols after P2 gel chromatography was further separated on aminopropyl-functionalized silica gel. (FIG. 11). Column 4 was loaded with a similar amount of Fagopyritol A3, but the other fagopyritols contributed to large total amount on the column. Fagopyritols A2 and B2 remained mixed, but several “pure” Fagopyritols A1, B1, and A3 fractions were pooled for further purification.

Example 13 Fractionation on Carbon-Celite Columns

A mixture of crude extracts was prepared from three 25 g samples of buckwheat purified bran and contained a total of 1.2 g soluble carbohydrates, of which 540 mg were fagopyritols. Purified bran was extracted with 50% ethanol, the ethanol was removed by rotary evaporation, and the extract was then passed through columns containing polyvinylpolypyrrolidone (“PVPP”) and Amberlite IRN-150 ion exchange resin. The extract was then digested with yeast (2 mg yeast per g of purified bran) to remove the 97% of the sucrose. A by-product of this treatment was the disaccharide trehalose (O-α-D-glucopyranosyl-(1→1)-O-α-D-glucoside). The yeast treated extracts were filtered through a 10,000 MW cut-off filter (Amicon) and freeze dried. The combined extracts were then re-dissolved in 9 ml of distilled water and applied to the top of the carbon-Celite column. The column contained a mixture of carbon (“Darco-G60,” J. T. Baker, Phillipsburg, N.J.) and Celite 545-AW (Supelco, Bellefonte, Pa.) in equal proportions by weight. The carbon-Celite mixture was slurry packed in distilled water into a 3.3 cm, ED column to a height of 17 cm, and the packed column washed with 1 liter of distilled water. The column was eluted stepwise at 4° C. with 800 ml distilled water, 1000 ml 5% ethanol, 1000 ml 10% ethanol, 250 ml 20% ethanol, and 200 ml 50% ethanol. Eluate was collected in flasks which were changed by hand at hourly intervals. On average, the flasks contained a volume of 37 ml. The fagopyritol concentration contained in the fractions was analyzed by high resolution gas chromatography. The monosaccharides, glucose, and fructose (present in large quantities due to the yeast treatment) and myo-inositol and D-chiro-inositol eluted in the water fractions. Fagopyritol B1 was eluted from the column by 5% ethanol, but was contaminated with galactinol. Fagopyritols A1 and B2 were eluted by 10% ethanol, with contamination from trehalose and sucrose (the small amount not removed by the yeast), respectively. Fagopyritol A2 was contained in the 20% ethanol elution, and Fagopyritol A3 remained on the column until the 50% ethanol elution. The carbon-Celite column was more effective than the 3-aminopropyl-functionalized silica gel column in removing colored contaminants.

Example 14 Hot vs. Cold Extraction of Flour

Flour (passed 100 mesh sieve, C&N Laboratory Mill) from whole white groats (Birkett Mills) was extracted in various ratios of solvent to flour and with hot extraction or cold extraction using a Waring blender equipped with a “high shear” polytron head. The blender was on 10 sec and off 10 sec for 1 or 2 min. After hot extraction, the mixture was too thick (due to swelling of the starch) to filter or centrifuge efficiently, resulting in low volumes of extracted fagopyritols. Cold extraction (either chilled or at room temperature) resulted in highest yields. Optimum extraction was with solvent/flour ratio of 20:1 (e.g., 25 g flour in 500 ml of ethanol:water (50:50)). Relative yields of fagopyritols were about 90%, 9%, and 1% for first, second, and third extraction of the same 25 g sample. The supernatant after centrifugation or filtrate after filtration of cold extract was heated to 80° C. to inactivate alpha-galactosidase and other enzymes present in the flour that might have hydrolyzed the fagopyritols during extraction. These enzymes were inactive in ethanol:water (50:50) but may have become activated in the water phase after the ethanol is evaporated during concentration.

Example 15 Effects of Using Defatted Flour

Some samples were defatted with hexane before extraction. In these experiments, the PVP column and ion exchange column steps were carried out after ultrafiltration. Although the early steps seemed to go easier, ultrafiltration was still slow, making the whole procedure was very time consuming and laborious. To simplify the procedure and improve efficiency, further experiments were carried out with non-defatted flour.

Example 16 Extraction with Water vs. Ethanol:water (50:50)

Three replicate samples of Minn-Dak flour (200 mg each) were extracted with 2 ml of distilled water or 2 ml of ethanol:water (50:50) in centrifuge tubes. Yield of fagopyritols was the same after extraction with water or ethanol:water (50:50) (Table 9). The water extracts appeared cloudy and contained more suspended materials. This observation was probably related to the higher solubility of proteins and starch in water. The ethanol extracts contained less suspended materials and also minimized microbial and endogenous enzyme activities during extraction. Ethanol:water (50:50) was used in subsequent extractions.

TABLE 9 Distilled Water Extraction Ethanol:water (50:50) Extraction 1 2 3 4 1 2 3 Mean Soluble Carbohydrate gg/200 mg flour gg/200 mg flour glucose 0.18 0.22 0.29 0.23 0.36 0.31 0.28 0.32 D-chiro-inositol 0.21 0.31 0.19 0.24 0.25 0.19 0.30 0.25 myo-inositol 0.03 0.04 0.02 0.03 0.02 0.02 0.04 0.03 sucrose 5.65 7.10 5.78 6.18 7.22 5.75 7.28 6.75 Fagopyritol A1 0.42 0.61 0.43 0.49 0.55 0.42 0.58 0.52 Fagopyritol B1 2.61 3.70 2.59 2.97 3.33 2.52 3.54 3.13 Fagopyritol A2 0.20 0.31 0.25 0.25 0.33 0.21 0.31 0.28 Fagopyritol B2 0.23 0.31 0.28 0.27 0.33 0.22 0.01 0.19 Fagopyritol A3 0.00 0.00 0.20 0.07 0.19 0.00 0.00 0.06 Total 9.53 12.60 10.03 10.72 12.58 9.64 12.34 11.52

Example 17 Comparison of Birkett Mills and Minn-Dak Flours

Sucrose and Fagopyritol B1 were the major components in both sources (Table 10). The Birkett Mills source was from whole groats and yielded about two times as much total soluble carbohydrates than the more refined flour of the Minn-Dak source. Sucrose was about 60% of the total soluble carbohydrates in both sources. Extracts from the Birkett Mills source were reddish brown in contrast to the cloudy white color the Minn-Dak extracts. Fagopyritols A2, B2, and A3 were not detected in this sample of Minn-Dak flour.

TABLE 10 Birkett Mills Minn-Dak Soluble mg/25 g mg/25 g carbohydrate flour % of total flour % of total D-chiro-inositol 11.2 2.6 5.7 2.9 myo-inositol 8.4 1.9 3.9 2.0 sucrose 250.0 57.1 120.3 60.8 Fagopyritol A1 22.9 5.2 11.6 5.9 Fagopyritol B1 123.3 28.2 56.4 28.5 Fagopyritol A2 9.2 2.1 0.0 Fagopyritol B2 10.2 2.3 0.0 Fagopyritol A3 2.6 0.6 0.0 Total 437.8 100.0 197.9 100.0

Example 18 Yeast Digestion of Sucrose and Fagopvritols

A 25 g sample of Minn-Dak flour was extracted with 500 ml of ethanol:water (50:50), centrifuged, heated to evaporate ethanol, and filtered. Cake yeast (8500 mg) was added to the aqueous extract and incubated at 22° C. for 0 to 48 h. About 97% of the sucrose was digested within the first 6 h, but some fagopyritols were lost, and D-chiro-inositol increased (Table 11). The large increase in myo-inositol was surprising and probably reflected a degradation of phytin in the flour. It is believed that better results could be obtained by removing the phytin with an ion exchange resin before digesting with yeast. Compared to sucrose, the fagopyritols were digested by yeast much more slowly.

TABLE 11 Soluble Hours Incubation Carbohydrate 0 6 20 29 42 44 D-chiro-inositol 4.4 7.6 15.0 15.6 14.9 27.2 myo-inositol 1.2 15.9 28.9 22.2 14.7 24.9 Sucrose 100.9 3.0 1.8 3.8 4.2 0.0 Fagopyritol A1 8.8 8.4 8.9 7.2 5.0 9.8 Fagopyritol B1 52.0 38.6 35.0 26.2 16.6 31.3 Total 188.1 73.5 89.6 75.0 55.4 93.2

In another yeast digestion experiment, the extract was passed through a PVP column, an ion exchange column, and an Amicon ultrafiltration apparatus before adding yeast. This experiment was carried out using dry granular yeast (200 mg). The results showed that sucrose was digested within the first 6 h, during which time the fagopyritols appeared to be relatively stable (Table 12). An initially unknown material increased during the first six hours and then slowly degraded. This unknown was identified as trehalose by cochromatography with authentic trehalose (after derivitization) and by gas chromatography/mass spectroscopy. Retention times on the DB1 and DB5 columns and mass spectra of the unknown were identical to those of authentic trehalose. α,α-Trehalose is a dimer of glucose (O-α-D-glucopyranosyl-(1-1′)-O-α-D-glucoside).

TABLE 12 Hours Incubation Soluble Carbohydrate 0 6 12 24 30 36 48 54 60 72 D-chiro-inositol 3.9 3.5 3.6 4.5 4.6 4.4 3.9 3.6 5.4 4.4 myo-inositol 2.6 2.3 2.4 2.8 3.3 2.9 2.6 2.1 3.2 2.3 sucrose 53.9 trehalose 0.5 22.4 16.0 11.3 10.0 6.3 3.8 3.4 3.7 2.4 Fagopyritol A1 6.2 5.9 5.6 7.0 6.7 7.3 5.8 5.3 6.3 4.7 Fagopyritol B1 36.7 32.5 33.1 37.0 40.2 40.9 34.9 31.5 37.7 29.3 galactinol 0.5 Fagopyritol A2 1.3 1.3 1.7 1.8 2.8 2.4 1.3 1.4 0.9 0.4 Fagopyritol B2 2.3 1.7 1.6 1.6 0.5 3.4 1.9 3.0 1.6 1.0 Total 107.9 69.6 64.0 66.0 68.1 67.6 54.2 50.3 58.8 44.5

Lower quantities of yeast were tested in a third experiment using the Birkett Mills source of flour. About 97% of the sucrose was digested by 50 mg of yeast within the first 2 h and accompanied by a doubling of glucose (Table 13). Fagopyritols appeared to be relatively stable during incubation with yeast. The lower amounts of yeast are preferred since the trehalose produced is also at a lower level. It is probably easier to remove the monosaccharide than higher molecular weight contaminants from fagopyritols.

TABLE 13 50 mg yeast 100 mg yeast 0 h 2 h 4 h 6 h 0 h 2 h 4 h 6 h Soluble Carbohydrate mg/25 g flour mg/25 g flour glucose 7.9 17.4 9.7 3.0 7.4 10.0 3.4 1.0 D-chiro-inositol 3.8 4.5 4.8 4.8 3.4 3.5 3.6 3.4 myo-inositol 1.3 1.4 1.6 1.1 1.1 1.1 1.1 1.3 sucrose 35.8 0.9 31.6 trehalose 2.9 2.8 2.9 1.0 5.2 4.5 4.5 Fagopyritol A1 7.1 8.1 8.1 8.2 6.3 6.4 6.3 6.0 Fagopyritol B1 44.4 49.7 48.7 51.0 38.4 38.9 38.0 38.8 galactinol 0.8 0.9 0.8 0.7 0.4 0.7 Fagopyritol A2 4.9 5.3 4.6 4.6 3.6 3.7 3.6 4.6 Fagopyritol B2 6.3 6.5 5.5 6.2 4.6 4.6 4.5 4.6 digalactosyl myo-inositol 1.1 1.1 1.1 0.6 0.5 Fagopyritol A3 2.7 2.8 2.3 2.3 2.6 Total 116.1 101.5 85.8 86.5 101.0 74.3 65.0 67.5 % of original 100.0 87.4 73.9 74.5 100.0 73.6 64.4 68.8

Example 19 Purification Steps

Table 14 shows the results of fagopyritol analyses at different stages (extraction, heat and centrifugation, and evaporation) during isolation from two twice-extracted 25 g samples of Birkett Mills whole groat buckwheat flour. During successive steps of purification, higher molecular weight fagopyritols appeared easier to resolve. This is believed to be partly due to increasingly pure samples and partly due to more concentrated samples in which Fagopyritol A3 is more easily resolved. The results indicated that shortening the extraction procedure may also be desirable With cold extraction, one can use smaller volumes and still obtain good yields of extracts.

TABLE 14 Extraction Heat & centrifugation Evaporation Soluble #1 #2 #3 #4 #5 Carbohydrate mg/25 g whole groat buckwheat flour D-chiro-inositol 11.2 9.4 9.3 9.2 9.8 myo-inositol 8.4 7.0 6.7 6.6 6.3 sucrose 277.6 252.2 254.0 245.6 240.6 Fagopyritol A1 22.9 20.7 19.2 19.7 19.6 Fagopyritol B1 123.4 185.6 103.7 111.8 106.2 Fagopyritol A2 9.2 11.0 10.1 11.7 11.7 Fagopyritol B2 10.2 11.9 10.5 14.5 11.5 Fagopyritol A3 7.9 9.2 6.8 Total 463.0 497.7 421.4 428.3 412.4 #1 and #2 by 0.5 ml samples taken from two 650 ml extracts immediately after extraction. #3 and #4 by 0.5 ml samples taken from two 350 ml extracts after heating and centrifugation. #5 by 0.5 ml samples taken from 250 ml extract after evaporation and centrifugation.

The amount of fagopyritols extracted by first and second extractions are presented in Table 15. Whole groat buckwheat flour (25 g, Birkett Mills) was extracted with 320 ml of ethanol:water (50:50) during first extraction and with 200 ml of ethanol:water (50:50) during the second extraction. As Table 15 shows, the first extraction removed about 90% of the soluble carbohydrates. The second extraction removed more Fagopyritol A1 but not detectable levels of the larger fagopyritols. These results indicate that shortening the extraction to a single step with lower volume would reduce extraction time but may not fully recover Fagopyritol A1.

TABLE 15 First Extraction Second Extraction Soluble mg/25 g % of mg 25 g % of Carbohydrate flour total flour total Total glucose 10.0 77 3.0 23 13.0 D-chiro-inositol 12.0 86 1.9 14 13.9 myo-inositol 5.0 59 3.5 41 8.5 sucrose 264.0 92 22.0 8 286.0 Fagopyritol A1 2.0 45 2.4 55 4.4 Fagopyritol B1 130.0 92 11.4 8 141.4 Fagopyritol A2 14.0 100 0 14.0 Fagopyritol B2 14.0 100 0 5.0 Fagopyritol A3 5.0 100 0 5.0 Total 456.0 91 44.2 9 500.2

The crude fagopyritol extracts could be separated into dimers, trimers, and tetramers on the 3-aminopropyl-functionalized silica gel column (FIG. 12). The column was overloaded in order to recover Fagopyritol A3. In spite of the overloading, considerable (but not total) purification was achieved. It was noted that the Fagopyritol A's and Fagopyritol B's usually overlap when the column is overloaded. However, with care, one can obtain relatively pure fractions even when the column is overloaded (FIG. 13).

Placing the PVP column before the ultrafiltration equipment hastened the passage of extracts through the Amicon 10,000 M_(r) cut-off filter. As indicated by FIGS. 14 and 15, this also improved the resolution between dimer, trimer, and tetramer fagopyritols.

Example 20 In Vito Fermentation of Fagopyritol-Enriched Buckwheat Extract By Human Fecal Microflora

Fermentation tests were done with fagopyritol-enriched Birkett Mills buckwheat extract that had previously been treated with dry granular baking yeast. Yeast cells were removed by centrifugation (10 minutes at 19K rpm using a Sorvall refrigerated centrifuge) and ultrafiltration (Amicon filtration device, 10, 000 MW cut-off). The fagopyritol water extract was boiled under carbon dioxide to sterilize and remove all other gases.

Anaerobic conditions were maintained during the preparation of fecal inoculum and of all samples by working under streams of carbon dioxide gas. Fermentation bottles were sealed with rubber stoppers and metal caps.

Fagopyritol substrate was prepared first. Then, 1 ml was placed in all bottles except blanks (duplicate samples plus one control and one blank done for 0, 6, 12, and 24 hours). Next, the nutritive medium was prepared. Control bottles received 1 ml of the medium in place of inoculum. The remaining medium was used to dilute fresh feces 1:12, w/v, prepared as described in Barry et al., “In Vitro Fermentation of Neutral Monosaccharides by Ruminal and Human Fecal Microflora,” Acta Vet Scand, 86 (supplement): 93-95 (1989) and Guillon et al., “Characterization of Residual Fibers from Fermentation of Pea and Apple Fibres by Human Fecal Bacteria,” J. Sci. Food Agric., 68:521-529 (1995), which are hereby incorporated by reference. All samples and blanks were inoculated with 1 ml of fecal inoculum by syringe through the rubber stoppers.

A 40° C. water bath was used for the fermentation which was stopped at the specified times by opening bottles to air and immediately freezing.

The samples were prepared for GC analysis after thawing by centrifuging (microfuge, 8000 rpm) once and then again in 10,000 MW cut-off microfuge tubes. The salts present in those samples containing nutritive medium were removed by passing each sample through Amberlite mixed-bed resin, because salts interfere with TMS-derivitization. Two control samples of the original extract were also passed through the resin to insure that fagopyritols were not removed by the treatment.

A small volume of each (750 μL) was dried with 100 μg of internal standard (phenyl α-D-glucopyranoside) in silyation vials under a stream of nitrogen gas; vials were then placed in a desiccator over P205 overnight.

Trimethylsilyl derivitives were prepared by adding 100 μL of 1:1 pyridine:TMS solution and heating at 70° C. for 30 minutes.

Finally, derivitized samples were analyzed on the GC (HP 5890 Series II; DB-1 column; 0.25 mm i.d., 15 m length, 0.25 μm film thickness).

Fecal bacteria removed all fagopyritol from the samples. Inositol compounds and trehalose were also metabolized by the bacteria. Two peaks (with retention times at about 3.1 and 3.4) repeated on all sample and control chromatograms indicating a by-product of the yeast treatment present in the buckwheat extract that was not metabolized by fecal bacteria; these same peaks showed in the analysis of the original extract.

The internal standard showed in all analyses indicating successful derivitization. Table 16 shows the concentrations (reported in mg/750 μL) of inositols and fagopyritols in the controls and samples.

TABLE 16 Controls Samples 0 hrs 6 hrs 12 hrs 0 hrs 6 hrs 12 hrs D-chiro-inositol 99 102 58 47 0 0 myo-inositol 45 48 26 20 0 0 Fagopyritol A1 88 87 94 106 0 0 Fagopyritol B1 667 636 605 591 0 0 Fagopyritol A2 56 54 59 54 0 0 Fagopyritol B2 83 72 66 65 0 0 Fagopyritol A3 21 20 26 48 0 0

Example 21 Procedure for Pilot Plant Production of Fagopyritols

Milling. Buckwheat bran samples were ground using an Alpine grinder (Augsberg, Germany) with the settings on slow speed and fast feed. Ten samples of 50 pounds (22.6 kg) of bran were ground, each sample taking approximately 1.5 hours to complete, resulting in an alteration in particle size.

Extraction. Ground bran samples were placed in a 100-gallon steam-jacketed vat with a built-in air-pressure driven stirrer (Lee Metal Products, Philipsburg, Pa.). The bran samples were extracted with 66 gallons of 50% ethanol (35 gallons 95% ethanol and 31 gallons distilled water) and stirred at maximum speed for 1 hour at room temperature. The mixture was allowed to settle for 1 hour, and, once settled, the supernatant was siphoned off through a plastic hose into two 30-gallon stainless steel drums on wheels.

For runs 1 and 2 the solids remaining were then rinsed by adding a further 15 gallons 50% ethanol (8 gallons 95% ethanol and 7 gallons distilled water). This was allowed to settle, and the supernatant siphoned off. For Runs 3 to 10 the rinsing step was omitted since the technique for removing the supernatant from the first extraction was improved. This involved first filtering the clear supernatant through a diatomaceous earth plate filter (“DEPF”) and then filtering as much of the sludge at the bottom of the vat as possible before the plate filter clogged.

Evaporation. The combined supernatant in Run 1 was evaporated at 60° C., with a 20 psi vacuum, in a 20-gallon capacity drum-vacuum steam-kettle (Goren, Chicago, Ill.). However, a substantial proportion of the extract was lost due to frothing up through the vacuum exhaust pipe.

In Run 2, the ethanol was evaporated by heating in a 100-gallon steam-jacketed vat. This method was effective, though time consuming (taking one full day), and resulted in ethanol fumes being released in the pilot plant.

A trial was performed with 7 gallons of the extract from Run 3 using the 20-gallon capacity drum vacuum steam kettle for evaporation, but with the exhaust hose fitted with a piece of plexiglass so that any loss of the extract could be visualized and therefore reduced by adjustment of temperature. The process was slow, as the temperature had to be lowered to 50° C. to prevent excessive frothing of the sample, and so the extract was evaporated only to 17% ethanol.

The rest of Run 3, and Runs 4, 5, and 6, were each concentrated using a Unipektin concentrator (Unipektin AG, Zurich, Switzerland). This vacuum-assisted process allowed the temperature to be maintained at 35-40° C. and operated by increasing the surface area of the extract by running it through a series of long, thin pipes. Although the Unipektin was reasonably fast to use, maintaining a low temperature did not reduce the formation of the muddy-brown color, and even passing each extract through the concentrator 4 times only reduced the ethanol concentration to 5.5%. The ethanol content of the extracts was reduced to 1 to 2% by boiling in a steam jacketed 25-gallon vat (Lee Metal Products, Philipsburg, Pa.).

For the evaporation of Runs 7 to 10, the 100-gallon steam-jacketed vat was used, as described for Run 2, with ethanol vapors discharged into the pilot plant. Ethanol concentration was measured using a model 360 ebulliometer (Dujardin-Sallerson, Paris, France).

Storage of Extracts. The extracts containing fagopyritols in ethanol (before evaporation) were stored at +2° C., and the aqueous extracts (after the evaporation step) were stored at −20° C. Extracts were stored in plastic 4 or 5-gallon containers with lids.

Diatomaceous Earth Plate Filter. The diatomaceous earth plate filter (“DEPF”) (T. Shriver Co., Harrison, New Jersey) consists of 6 metal plates (7 inches×7 inches) with a plastic holder and piece of filter paper (Filtration Unlimited, Akron, N.Y.) on either side, all squeezed tightly together. The DEPF was prepared for use by feeding through 120 g (20 g per plate) of pre-coat mixed in water; when the water passing through the DEPF was clear, the pre-coat was in place. Body feed was stirred into the fagopyritol extract. The sample with body feed was then passed through the DEPF, with continuous stirring to keep the body feed in suspension. The diatomaceous earth powders used as pre-coat or body feed were Celite 503, Celite 535, Celite 577, and Hi-flo Super Cel (“HFSC”) (Celite Corp., Lompoc, Calif.). Unless stated otherwise, the combination used was Celite 577 as pre-coat (20 g per plate) and HFSC as body feed (0.25% weight of dry powder to weight of extract).

PVPP Treatment. The polyvinylpolypyrrolidone (PVPP) used in the pilot plant extractions was Polyclar-VT (ISP Technologies, Wayne, N.J.). Where PVPP treatment was performed after the extract had been concentrated by evaporation, 0.75 kg PVPP (unless stated otherwise) was added to the extract in a 25-gallon steam-jacketed vat and stirred for 30 minutes with an air pressure-driven stirrer (Gast, Benton Harbor, Mich.). This removed some coloration but was not as effective as it had been on a laboratory bench-scale where the PVPP was loaded into a column and the extract passed through it. Increasing the treatment time to 3 hours (Run 2), as used in the wine industry, did not noticeably improve the resulting color of the extract.

For runs 7 to 10, 1.5 kg PVPP was added to the ethanolic extract of fagopyritols in the 100-gallon vat prior to evaporation. The mixture was stirred for 30 minutes and allowed to settle for a further 30 minutes, and the supernatant removed. After evaporation, the extract became a deep yellow color (soluble proanthocyanidins) rather than muddy brown (oxidized and condensed phenolics). The 0.75 kg PVPP treatment described above was also used after evaporation to further clean the extracts. Runs 7 to 10 were consequently much cleaner than the other extracts with regard to colored contaminants.

Initially, the PVPP was allowed to settle, the supernatant removed for DEPF, and the PVPP and associated liquid at the base discarded. Subsequently, it was found that the whole extract, including PVPP, could be fed into the DEPF, the PVPP being retained by the DEPF without clogging.

Bentonite Treatment. Bentonite is used in the wine industry to clear haze caused by unstable proteins in wine reacting with proanthocyanidins. The bentonite (Presque Isle Wine Cellars, North East, Pa.) was prepared by mixing (3 g per gallon of sample unless stated otherwise) with a small amount of distilled water to form a thick slurry. A blender was used to dissolve lumps. After 2 to 3 hours, a small amount of extract was mixed with the bentonite slurry, and then this was added to the rest of the extract and stirred for a few minutes. The treatment was allowed to run for approximately 24 hours in cold storage to allow flocculation to occur and proteins to precipitate. The supernatant was carefully poured off. In contrast to PVPP, the use of DEPF for removing the bentonite was not suitable, because the DEPF clogged easily.

Ion Exchange Treatment. Syburn resin (Aftech, Rochester, N.Y.) was added (1.8 kg) and stirred for 30 minutes. The resin was removed from the sample by passing through a 100-mesh sieve.

Yeast Treatment. Treatment of the extract with yeast to remove sucrose was performed for Run 2. The extract was warmed to 32° C., and 40 g of dry granular baking yeast was added and allowed to ferment for 1 hour with occasional stirring. The yeast was killed by heating to 77° C. for 15 minutes. This practice was discontinued because, although the yeast was efficient at removing the sucrose, it produced trehalose, which was difficult to separate from Fagopyritol A1 during subsequent purification of fagopyritols.

Ultrafiltration. Two 10,000 MW cut-off hollow fiber filter cartridges were fitted to an ultrafiltration unit (“UF”) (Romicon, Wilmington, Mass.).

Run 1 was passed through the UF early in the extraction process, quickly filtering the large volume of ethanolic extract. It was clear from this run that a large volume of liquid should be used with the UF as there was a void volume of about 5 gallons. Also, the UF should be used after most of the ethanol has been removed from the extract as the filter membranes curled slightly in the presence of 50% ethanol, though were still re-usable. Both filtration and cleaning the filters after use was found to be time and labor-intensive, so, to reduce labor costs, the other extracts were combined for their final UF treatment, rather than running each through individually.

Accordingly, extracts from Runs 2 to 10 were combined for ultrafiltration. The temperature was maintained at 27° C. throughout the 1.5 hours it took to filter the 65 gallons of total extract. Particles smaller than 10,000 daltons passed through the filter, whilst larger particles passed straight up through the cartridge and re-circulated. About 5 gallons of waste was accumulated which contained the large particulates. The filtered extract, which was collected in the 100-gallon vat, was clear and light yellow in color. The color probably reflects the presence of soluble, small molecular weight proanthocyanidins that could not be filtered by ultrafiltration and had escaped being adsorbed to PVPP, bentonite, or ion exchange resin, especially from Runs 2 to 6.

Example 22 Purification and Molecular Structure by NMR of Fagopyritol A2. Fagopvritol A3, and Fagopyritol B2.

Extraction of fagopyritols from Buckwheat. The bran milling fraction of common buckwheat (Fagopyrum esculentum Moench cv. Manor) seed was provided by Minn-Dak Growers, Ltd. (Grand Forks, N.Dak.). Buckwheat bran was extracted at pilot plant scale (see Example 21) with EtOH:H₂0 1:1 (v/v), the extract was filtered through a diatomaceous earth plate filter, evaporated to reduce the volume and remove EtOH, and treated with polyvinylpolypyrrolidone (PVPP), bentonite, and ion exchange resin to remove non-carbohydrate components. The soluble carbohydrate extract was finally filtered through 10,000 MW cut-off hollow fiber filter cartridges and evaporated to reduce the volume.

Purification ofFagopyritols. Purification was performed by preparative chromatography on a stationary phase of carbon (Darco G60; J. T. Baker, Phillipsburg, N.J.) and Celite 545-AW (Supelco, Bellefonte, Pa.), 1:1 (w/w) (Whistler et al., J. Amer. Chem. Soc., 72:677-679 (1950), which is hereby incorporated by reference). Columns were freshly slurry packed in water, freeze-dried samples were dissolved in minimal water for loading, and soluble carbohydrates were eluted at 4° C. with stepwise increments of EtOH:H₂0 (v/v) and collected in 20-mL fractions. Samples of column eluate were taken periodically, phenyl α D-glucoside was added as internal standard, dried samples were derivatized with N-trimethylsilylimidazole:pyridine, 1:1 (v/v), and analyzed (Horbowicz, which is hereby incorporated by reference) on a Hewlett Packard 6890 GC equipped with a flame ionization detector, split-mode injector (1:50), and an HP-1MS capillary column (15 m×0.25 mm i.d., 0.25 μm film thickness). The GC was operated with a programmed initial temperature of 1 50° C., adjusted to 200° C. at 3° C./min, adjusted to 325° C. at 7° C./min, and held at 325° C. for 20 The injector port was operated at 335° C. and the detector at 350° C. The carrier gas was nitrogen at 2.5 mL/min.

For purification of Fagopyritol B2, Fagopyritol A2, and Fagopyritol A3, buckwheat bran extract was chromatographed on a 25×900 mm bed. Following successive elution with 2.2 L of water, 2.5 L of 4% EtOH, 3L of 5% EtOH, Fagopyritol B2 was eluted with 2L of 7% EtOH, Fagopyritol A2 with 1L of 12% EtOH, and Fagopyritol A3 with 1L of 20% EtOH. Fractions containing Fagopyritol B2 or Fagopyritol A2 or Fagopyritol A3 were freeze-dried and re-chromatographed.

Fractions of Fagopyritol B2 were treated with invertase (EC 3.2.1.26) from Candida utilis for 1 h at 22° C. to hydrolyze sucrose, and then chromatographed on a 15×450 mm bed. Following elution of contaminants with 1 L of 5% EtOH, Fagopyritol B2 was eluted with 6% EtOH.

Fagopyritol A2 was chromatographed on a 15×500 mm bed. Low levels of contaminants were eluted sequentially with 0.5L of H₂O, 1 L of 6% EtOH, and 0.5L of 8% EtOH, and Fagopyritol A2 eluted with 9% EtOH.

Fractions containing Fagopyritol A3 from four separate columns of buckwheat bran extract were combined and re-chromatographed (19×350 mm). Fagopyritol A3 was eluted with 14% EtOH following removal of contaminants by elution with 12% EtOH.

Acid and Enzymic Hydrolysis. Samples of Fagopyritol B2, Fagopyritol A2, and Fagopyritol A3 were hydrolyzed with IM CF₃CO₂H at 80° C. for 16 h and evaporated to dryness. Mole ratios of D-galactose and D-chiro-inositol were calculated after GC analysis of the trimethylsilyl-products. Samples (100 μg) of Fagopyritol B2, Fagopyritol A2, and Fagopyritol A3 were incubated with 1.25 units desalted green coffee bean α-D-galactosidase (EC 3.2.1.22) in 200 μL water at 22° C. or with 0.5 units bovine liver β-D-galactosidase (EC 3.2.1.23) in 200 μL water at 37° C. for 24 h. Enzyme protein was removed by filtration (10,000 MW cut-off filter), samples were dried, and products assayed by GC analysis of trimethylsilyl derivatives. Raffinose and lactose (100 μg) were hydrolyzed only by α- or β-D-galactosidase respectively, confirming that both enzymes were active.

Analysis of Chirality of Chiro-Inositol. Authentic D-chiro-inositol and L-chiro-inositol standards (Aldrich, Milwaukee, Wis.) and the acid hydrolysis products (2 M CF₃CO₂H, 70° C. for 3 h) of Fagopyritol B2, Fagopyritol A2, and Fagopyritol A3 were derivatized with pentafluoropropionic anhydride:acetonitrile (1:1, v/v) (Ostlund et al., Proc. Natl. Acad. Sci. USA, 90:9988-9992 (1993)). Derivatized products were co-chromatographed with the derivatized standards by GC using a Chirasil-Val chiral capillary column (25 m×0.25 mm i.d., 0.16 μm film thickness) (Leavitt et al., Carbohydr. Res., 103:203-212 (1982)) run at 100° C. isothermally on a Hewlett Packard 5890 Series 11 GC. Injection port temperature was 130° C. and FID temperature was 130° C. Helium was the carrier gas at 3 mL/min, split 1:50.

NMR Analysis. Purified samples (41 mg of Fagopyritol B2, 13.5 mg of Fagopyritol A2, 34.5 mg of Fagopyritol A3) were dissolved in 700 μL of 99.96% D₂O (Cambridge Isotope Labs, Andover, Mass.) for analysis. A Varian (Palo Alto, Calif.) VXRS-400 was used at 100.5 MHz for iD 13C NMR and DEPT (distortionless enhancement by polarization transfer) at 25° C., with 1,4-dioxane (δ_(c) 67.4) as external carbon standard. All other NMR experiments were performed at 499.9 MHz on a Varian Unity-500, standardized against the residual HOD (δ_(H) 4.63) at 25° C. These were 1D ¹H NMR, double-quantum filtered correlated spectroscopy (DQCOSY), gradient-enhanced heteronuclear multiple quantum correlation (GHMQC), homonuclear 2D J-spectroscopy (HOM2DJ), Nuclear Overhauser and Exchange Spectroscopy (NOESY), and gradient-enhanced heteronuclear multiple bond correlation (GHMBC). Data were analyzed and simulations performed using Varian VNMR software, version 6.1A.

Results. Buckwheat bran was extracted with 50% EtOH and fagopyritols were purified using column chromatography with carbon-Celite, 1:1 (w/w). Fagopyritol B2 was purified from fractions eluted with 7% EtOH, Fagopyritol A2 was eluted with 9% EtOH, and Fagopyritol A3 was eluted with 14% EtOH. Per(pentafloroproprionates) of chiro-inositol from acid hydrolysis of Fagopyritol B2, Fagopyritol A2, and Fagopyritol A3 co-chromatographed on a chiral column with D-chiro-inositol but not with L-chiro-inositol. Acid hydrolysis of Fagopyritol B2, Fagopyritol A2, and Fagopyritol A3 produced D-galactose and D-chiro-inositol in mole ratios of 2.02:1.00 for Fagopyritol B2, 2.03:1.00 for Fagopyritol A2, and 2.98:1.00 for Fagopyritol A3. GC analysis of partial hydrolysis products of Fagopyritol B2, Fagopyritol A2, and Fagopyritol A3 identified Fagopyritol B1 from Fagopyritol B2, Fagopyritol A1 from Fagopyritol A2, Fagopyritol A2 and Fagopyritol A1 from Fagopyritol A3, D-chiro-inositol, and galactose from Fagopyritol B2, Fagopyritol A2, and Fagopyritol A3. Fagopyritol B2, Fagopyritol A2, and Fagopyritol A3 were hydrolyzed by α-D-galactosidase, but not by β-D-galactosidase, demonstrating α-anomeric linkages for all D-galactopyranosyl residues.

¹³C NMR spectra for Fagopyritol B2 identified 18 carbon resonances, and ¹H NMR spectra identified 20 hydrogen resonances. Chemical shifts for the 18 carbons were each linked to those of the 20 hydrogens using GHMQC. All but four of the carbon chemical shifts were between 69 and 76 ppm. Two carbon resonances had chemical shifts downfield at 99.02 and 95.74 ppm, characteristic of substituted anomeric carbons. Two carbons were upfield at 62.03 and 67.72 ppm, and these were identified as CH₂ carbons by DEPT. All but 3 of the hydrogens had chemical shifts between 3.5 and 3.9 ppm. The two most downfield hydrogens (δ 4.81 and δ 4.99) had chemical shifts characteristic of anomeric hydrogens, and GHMQC showed attachment to anomeric carbons (δ 99.02 and δ95.74, respectively). These served as the starting points for tracing connectivity within the galactose spin systems by DQCOSY. Assignment of hydrogen resonances to the upfield CH₂ carbons allowed distinction between the two galactose residues; one of the CH₂ carbons (δ 62.03) had two hydrogens with accidentally equivalent resonances (δ 3.59), while hydrogens on the other CH₂ carbon (δ 67.72) had non-equivalent resonances (δ 3.72 and δ 3.54). The presence of non-equivalent resonances between CH₂ hydrogens is suggestive of further substitution at this carbon; thus, the spin system involving the non-equivalent hydrogens (δ 3.54, H-6′a; δ 3.72, H-6′b) was assigned to the galactose proximal (closest) to chiro-inositol, and the spin system involving the equivalent protons (δ 3.59, H-6″a, H-6″b) was assigned to the distal galactose residue (Table 17).

TABLE 17 ¹H and ¹³C NMR (25° C., D₂O) chemical shifts and proton-proton coupling constants of Fagopyritol B2 (1) and Fagopyritol A2 (2) Chemical Shifts (ppm) Coupling Constants (Hz) 1 2 1 2 Position (no.) δ_(H) δ_(C) δ_(H) δ_(C) (no.) J (Hz) J (Hz) Cyclitol ring 1 4.08 68.14 3.86  72.57 J_(1,2)  3.0  3.2 2 3.73 75.58 3.67  70.14^(a) J_(2,3) 10.0  9.8 3 3.59 71.97 3.54  81.78 J_(3,4)  9.6  9.8 4 3.48 73.59 3.61  73.65^(b) J_(4,5)  9.8  9.8^(c) 5 3.64 71.25 3.61  71.05^(b) J_(5,6)  3.2  3.2 6 3.90 71.76 3.83  72.12 J_(6,1)  4.1  4.0 Galactopyranosyl ring (proximal) 1′ 4.99 95.74 5.14 100.33 J_(1′,2′)  3.9  3.9 2′ 3.72 69.10^(d) 3.67  69.45 J_(2′,3′) 10.4 10.4 3′ 3.82 70.21^(e) 3.75  70.04 J_(3′,4′)  3.4  3.2 4′ 3.90 70.13 3.86  69.93 J_(4′,5′)  1.2  1.3 5′ 4.25 69.68 4.30  70.06^(a) J_(5′,6a′)  4.4  4.9 6′a 3.54 67.72 3.51  67.53 J_(5′,6b′)  8.1  7.8 6′b 3.72 3.70 J_(6a′,6b′) 10.7 10.5 Galactopyranosyl ring (distal) 1″ 4.81 99.01 4.81  99.30 J_(1″,2″)  3.9  3.9 2″ 3.65 69.08^(d) 3.63  69.12 J_(2″,3″) 10.4 10.3 3″ 3.73 70.24^(c) 3.73  70.04 J_(3″,4″)  3.2  2.9 4″ 3.79 70.13 3.79  69.93 J_(4″,5″)  1.2  1.2 5″ 3.86 71.71 3.78  71.84 J_(5″,6a″)  6.2  6.1 6″a 3.59 62.03 3.56  62.02 J_(5″,6b″)  6.2  6.1 6″b 3.59 3.56 ^(a,b,d,e)Assignment may be reversed ^(c)Expected value; confirmed by simulation of experimental data

Additional evidence for this assignment came from C-6′ (δ 67.72) which is deshielded by 5.5 ppm in comparison to C-6″ (δ 62.03), and the adjacent C-5′ (δ 69.98) is shifted upfield by a small amount in comparison to C-5″ (δ 71.71), indicative of substitution at C-6′. The small coupling constants (J_(1′,2′), 3.9 Hz; J_(1″,2″)3.9 Hz) calculated from the ID ¹H spectra (Table 17) verified the a-anomeric linkage for both galactosyl rings.

For Fagopyritol B2, the six remaining ¹H resonances, all between 3.47 and 4.08 ppm, were assigned to D-chiro-inositol, and connectivity delineated from the DQCOSY experiment. The carbon to which each hydrogen was attached was estimated from GHMQC. The most downfield of these carbons (δ 75.58) in the cyclitol ring was the point of linkage to galactose. Coupling constants (J_(1,2) 3.0 Hz; J_(2,3) 10.0 Hz) observed for the hydrogen (δ 3.73, H-2) attached to the substituted carbon (δ 75.58) and those observed for adjacent hydrogens assigned H-1 (J_(1,2) 3.0 Hz; J_(1,6) 4.1 Hz) and H-3 (J_(2,3) 10.0 Hz; J_(3,4) 9.6 Hz), confirmed the assignment of 3.73 ppm to be an axial hydrogen with an axial and equatorial hydrogen adjacent (i.e., H-2 or H-5). Due to symmetry of the chiro-inositol ring, substitution at C-2 or C-5 results in equivalent structures, and the smaller number is assigned. Therefore, fagopyritol B2 is O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol.

¹³C NMR spectra for Fagopyritol A2 identified 18 carbon resonances, and ¹H NMR spectra identified 20 hydrogen resonances. The two galactosyl residues of Fagopyritol A2 were elucidated in the same way as described for Fagopyritol B2 (above), and the chemical shifts and coupling constants were comparable to those of Fagopyritol B2 (Table 17). For Fagopyritol A2, the hydrogen (δ 3.54) on the substituted carbon (δ 81.78) in the D-chiro-inositol ring was observed to have only large coupling constants (J_(2,3) 9.8 Hz; J_(3,4) 9.8 Hz) from both ¹H NMR and homonuclear 2D J-spectroscopy (“HOM2DJ”) experiments, indicating trans-axial vicinal hydrogens (i.e., H-3 or H-4). Due to symmetry of the chiro-inositol ring, substitution at C-3 or C-4 results in equivalent structures, and the smaller number is assigned. The point of linkage in the D-chiro-inositol residue was C-3 in Fagopyritol A2, as in Fagopyritol A1. The only apparent GHMBC interaction for C-1′ (δ 100.33) was with H-3 (δ 3.54), confirming the presence of axial hydrogens at H-2 and H-4 of the D-chiro-inositol ring. Similarly, the interaction between C-3 and H-1′ but not H-2′ confirmed the (1′→3) connectivity, with H-2′ being axial to the ring. Fagopyritol A2 is O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→3)-D-chiro-inositoI.

A clear differential can be seen between the fagopyritol A series and the fagopyritol B series in the chemical shift of the substituted carbon, which is the most downfield of the non-anomeric carbons. In the B series this is at 76 ppm, whereas for the A series the substituted carbon is 6 ppm further downfield at 82 ppm. This pattern defined Fagopyritol A3, a tri-galactosyl D-chiro-inositol, as a member of the fagopyritol A series, since it contains a carbon resonance at 82.04 ppm (C-3) (Table 18).

TABLE 18 ¹H and ¹³C NMR (25° C., D₂O) chemical shifts and ¹H coupling constants of Fagopyritol A3. Coupling Position Chemical Shifts (ppm) Constants (Hz) (no.) δ_(H) δ_(C) J Hz Cyclitol ring 1 3.91 72.56 J_(1,2) 2.9 2 3.73 70.11^(a) J_(2,3) 10.1 3 3.59 82.04 J_(3,4) 10.1 4 3.65 73.58^(b) J_(4,5) 10.1^(c) 5 3.65 71.06^(b) J_(5,6) 3.1 6 3.87 72.11 J_(6,1) 4.1 Galactopyranosyl ring (proximal) 1′ 5.20 100.36 J_(1′,2′) 4.0 2′ 3.72 69.45^(d) J_(2′,3′) 10.4 3′ 3.84 70.11^(a) J_(3′,4′) 3.3 4′ 3.88 70.03^(a) J_(4′,5′) 1.0 5′ 4.34 69.95 J_(5′,6′a) 4.1 6′a 3.50 67.61 J_(5′,6′b) 8.1 6′b 3.78 J_(6′a,6′b) 10.6 Galactopyranosyl ring (middle) 1″ 4.84^(c) 99.05 J_(1″,2″) 3.8 2″ 3.68 69.10 J_(2″,3″,) 10.4 3″ 3.79 70.18^(a) J_(3″,4″) 3.4 4″ 3.88 70.03^(a) J_(4″,5″) 1.0 5″ 4.02 69.53^(d) J_(5″,6″a) 4.8 6″a 3.57 67.26 J_(5″,6″b) 7.0 6″b 3.72 J_(6a″,6″b) 11.0 Galactopyranosyl ring (distal) 1′″ 4.85^(e) 98.85 J_(1′″,2′″) 3.8 2′″ 3.68 69.10 J_(2′″,3′″) 10.4 3′″ 3.79 70.23^(a) J_(3′″,4′″) 3.4 4′″ 3.72 70.11^(a) J_(4′″,5′″) 1.2 5′″ 3.87 71.74 J_(5′″,6′″a) 6.4 6′″a 3.60 61.95 J_(5′″,6′″b) 6.4 6′″b 3.60 ^(a, b, d, e)Assignment may be reversed ^(c)Expected value; confirmed by simulation of experimental data

Further, the hydrogen attached to the substituted carbon (δ 82.04) was observed to have a chemical shift of 3.59 ppm (H-3) with large coupling constants (J_(2,3) 10.1 Hz, J_(3,4) 10.1 Hz) calculated from ¹H NMR and HOM2DJ. This compound was previously predicted to be a tri-galactosyl D-chiro-inositol of the fagopyritol B series (Horbowicz and Horbowicz et al., Planta, 205:1-11 (1998), which are hereby incorporated by reference); since buckwheat seeds have more Fagopyritol B1 than Fagopyritol A1 and more Fagopyritol B2 than Fagopyritol A2, the same pattern was expected for tri-galactosyl D-chiro-inositols. However, this tri-galactosyl D-chiro-inositol was determined to be O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1−3)-D-chiro-inositol and assigned the trivial name Fagopyritol A3.

With four ring systems superimposed, spectra for Fagopyritol A3 were complex. Once most of the 27 hydrogens were identified from the 1D ¹H and HOM2DJ spectra, DQCOSY was used to assign hydrogens as belonging to galactose or D-chiro-inositol. The hydrogens at the 2, 3, and 4 positions of the three galactose rings had very similar or identical resonances between rings, making assignment of hydrogens into each individual ring not immediately apparent. Three pairs of hydrogens (δ 3.50 and δ 3.78; 8 3.57 and δ 3.72; δ 3.60) were identified as 6 position in the galactose residue by their interaction in the GHMQC experiment with the carbons (δ 67.61; δ 67.26; δ 61.95) identified as CH₂ by DEPT. One pair of hydrogens was accidentally equivalent (δ 3.60), linked to the most upfield carbon (δ 61.95), and assigned as H-6′″a and H-6′″b. These hydrogens (δ 3.60) interacted with the hydrogen at 3.87 ppm (H-5′″) by DQCOSY. Through its GHMBC interaction with H-5′″, the anomeric carbon at 98.85 ppm was assigned as C-1′″. This carbon (δ 98.85) also interacted with the CH₂ hydrogens with chemical shifts of 3.72 and 3.57 ppm, assigned H-6′″a and H-6″b. Similarly, the carbon at 99.05 ppm (C-1″) interacted with the hydrogen at 4.02 ppm (H-5″), and with hydrogens at 3.78 and 3.50 ppm (H-6′a and H-6′b). GHMBC interaction of C-1′ (δ 100.36) with only H-3 (δ 3.59) of D-chiro-inositol confirmed H-3 as attached to the carbon at the point of substitution (C-3), with axial H-2 and H-4. With these assignments made, the remaining assignments could be made for each of the four spin systems. Finally, GHMQC was used to assign carbons. With 8 of the 24 carbon resonances occurring between 69.95 and 70.23 ppm, absolute assignment was not possible, leading to a number of assignments being potentially reversible (Table 18).

It was possible to calculate the coupling constants between hydrogens in the D-chiro-inositol ring for the fagopyritol B series from their ID ¹H NMR spectra. However, in the fagopyritol A series chemical shifts of H-4 and H-5 coincide, and the strong coupling between H-4 and H-5 results in a complex multiplet in which it is not possible to find a coupling constant larger than 6.5 Hz. Simulation of the observed ¹H chemical shifts for D-chiro-inositol in Fagopyritol A2 or Fagopyritol A3 with coupling constants that would be expected for D-chiro-inositol based on those measured for the fagopyritol B series, results in a multiplet for H-4 and H-5 consistent with that observed in the fagopyritol A series. This confirmed the coupling constants (J_(4,5) 9.8 Hz) simulated for Fagopyritol A2 (Table 17) and (J_(4,5) 10.1 Hz) simulated for Fagopyritol A3 (Table 18).

Members of the fagopyritol B series have been previously identified in other species. In addition to buckwheat (Horbowicz et al., Planta, 205:1-11 (1998); Szczecinski et al., Bull. Pol. Acad. Sci. Chem., 46:9-13 (1998), which are hereby incorporated by reference), Fagopyritol B1 is in seeds of soybean (Schweizer et al., Carbohydr. Res., 95:61-71 (1981), which is hereby incorporated by reference), lupin, lentil, and chickpea (Quemener et al., Phytochem., 22:1745-1751 (1983), which is hereby incorporated by reference), and jojoba bean (Ogawa et al., Carbohydr. Res., 302:219-221 (1997), which is hereby incorporated by reference). Chemical synthesis of Fagopyritol B1 has been reported (Kornienko et al., Carbohydr. Res., 310:141-144 (1998), which is hereby incorporated by reference). Fagopyritol B2 was identified by chemical methods in seed balls of sugar beet (Shiomi et al, Agric. Biol. Chem., 52:1587-1588 (1988), which is hereby incorporated by reference), but its absolute configuration is first presented herein. Fagopyritol A2 and Fagopyritol A3, along with Fagopyritol A1, are members of a novel series of galactosyl cyclitols in buckwheat seeds.

Example 23 Purification and Molecular Structure by NMR of Fagopyritol A1 and Fagopyritol B1

Extraction ofFagopyritols from Buckwheat. The bran milling fraction of common buckwheat (Fagopyrum esculentum Moench cv. Manor) seed was provided by Minn-Dak Growers, Ltd. (Grand Forks, N.Dak.). Buckwheat bran was extracted at pilot plant scale (see Example 21) with EtOH:H₂O 1:1 (v/v), the extract was filtered through a diatomaceous earth plate filter, evaporated to reduce the volume and remove EtOH, and treated with polyvinylpolypyrrolidone (PVPP), bentonite, and ion exchange resin (mixed strong anionic and cationic) to reduce non-carbohydrate components. The soluble carbohydrate extracts were finally filtered through a 10,000 MW cut-off hollow fiber filter cartridge and evaporated to reduce the volume.

Purification ofFagopyritols. Purification was performed by preparative chromatography on a stationary phase of carbon (Darco G60; J. T. Baker, Phillipsburg, N.J.) and Celite 545-AW (Supelco, Bellefonte, Pa.), 1:1 (w/w) (Whistler et al., J. Amer. Chem. Soc., 72:677-679 (1950), which is hereby incorporated by reference). Columns were freshly slurry packed in water, freeze-dried samples were dissolved in minimal water for loading, and soluble carbohydrates were eluted at 4° C. with stepwise increments of EtOH:H₂O (v/v) and collected in 20-niL fractions. Samples of column eluate were taken periodically, phenyl α-D-glucoside was added as internal standard, dried samples were derivatized with N-trimethylsilylimidazole:pyridine, 1:1 (v/v), and analyzed (Horbowicz, which is hereby incorporated by reference) on a Hewlett Packard 6890 GC equipped with a flame ionization detector, split-mode injector (1:50), and an HP-1MS capillary column (15 m×0.25 mm i.d., 0.25 μm film thickness). The GC was operated with a programmed initial temperature of 150° C., adjusted to 200° C. at 3° C./min, adjusted to 325° C. at 7° C./min, and held at 325° C. for 20 The injector port was operated at 335° C. and the detector at 350° C. The carrier gas was nitrogen at 2.5 mL/min.

For purification of Fagopyritol A1, buckwheat bran extract containing approximately 1.8 g of soluble carbohydrates (5% Fagopyritol Al) was chromatographed on a 25×900 mm bed of carbon-Celite. Following elution with 2.2 L of water to remove monosaccharides, and 2.5 L of 4% EtOH to remove galactinol and Fagopyritol B1, Fagopyritol A1 was eluted with 5% EtOH, freeze-dried, and re-chromatographed (25 x 550 mm bed). Fagopyritol A1 (95% pure) was obtained by first removing Fagopyritol B1 with 4% EtOH, and then eluting Fagopyritol A1 with 5% EtOH. Fractions containing Fagopyritol A1 were re-chromatographed a third time (10 x 370 mm bed; eluted with water and then 5% EtOH) to obtain pure Fagopyritol A1 for NMR analysis.

For purification of Fagopyritol B1, buckwheat bran extract was incubated with baker's yeast to remove sucrose. A sample containing 3.15 g of soluble carbohydrates (25% Fagopyritol B1) was chromatographed on a 43 x 360 mm carbon-Celite bed. Following elution with 1.5 L water to remove monosaccharides, Fagopyritol B1 and galactinol were eluted with 4% EtOH. ,Fractions containing Fagopyritol B1 were freeze-dried and re-chromatographed on a 25 x 900 mm column. Galactinol co-eluted with the initial burst of Fagopyritol B1, allowing fractions from the tail of Fagopyritol B1 elution to be combined to give pure Fagopyritol B1 for NMR analysis.

D-chiro-inositol was obtained by hydrolysis of partially purified Fagopyritol B1 (106 mg of Fagopyritol B1; 14 mg of Fagopyritol A1; 2.5 mg of galactinol) with 4 M HCl for I h at 95° C., neutralized with NaOH, and the resulting mixture chromatographed on carbon-Celite (25 x 900 mm) and eluted with water.

Acid and Enzymic Hydrolysis. Samples of Fagopyritol A1 and Fagopyritol B1 were hydrolyzed with I M CF₃CO₂H at 80° C. for 16 h and evaporated to dryness. Mole ratios of D-galactose and D-chiro-inositol were calculated after GC analysis of the trimethylsilyl-products. Samples of Fagopyritol A1 and Fagopyritol B1 (200 g) were incubated with 1.25 units desalted green coffee bean a-D-galactosidase (EC 3.2.1.22) in 200 JAL water at 22° C. or with 0.5 units bovine liver P-D-galactosidase (EC 3.2.1.23) in 200 PLL water at 37° C. for 24 h. Enzyme protein was removed by filtration (10,000 MW cut-off filter), samples were dried, and products assayed by GC analysis of trimethylsilyl derivatives. Raffinose and lactose (100 jg) were hydrolyzed by oc-or P-D-galactosidase respectively, confirming that both enzymes were active.

Separation of D-chiro-inositol and L-chiro-inositol by Chiral Column GC. Authentic D-chiro-inositol and L-chiro-inositol standards (gift from A. Richter, University of Vienna) and the acid hydrolysis products (2 M CF₃CO₂H, 70° C. for 3 h) of pure Fagopyritol A1 or pure Fagopyritol B1 were derivatized with pentafluoropropionic anhydride: acetonitrile (1:1, v/v) (Ostlund et al., Proc. Natl. Acad. Sci. USA, 90:9988-9992 (1993), which is hereby incorporated by reference). Derivatized products were co-chromatographed with the derivatized standards by GC using a Chirasil-Val chiral capillary column (25 m×0.25 mm i.d., 0.16 μm film thickness) (Leavitt et al., Carbohydr. Res., 103:203-212 (1982), which is hereby incorporated by reference) run at 100° C. isothermally on a Hewlett Packard 5890 Series II GC. Injection port temperature was 130° C. and FID temperature was 130° C. Helium was the carrier gas at 3 mL/min, split 1:50.

NMR Analysis. Purified samples (14.4 mg of Fagopyritol A1; 21 mg of Fagopyritol B1; 9 mg of D-chiro-inositol) were dissolved in 700 μL of 99.96% D₂O for analysis. A Varian VXRS-400 was used for ID ¹³C NMR at 100.5 MHz, 25° C., with 1,4-dioxane (δ_(c) 67.4) as external carbon standard. All other NMR experiments were performed at 499.9 MHz on a Varian Unity-500 (Palo Alto, Calif.), standardized against the residual HOD (δ_(H) 4.63) at 25° C. These were 1 D ¹H NMR, double-quantum filtered COSY, gradient-enhanced HMQC, homonuclear 2D J-spectroscopy (HOM2DJ), NOESY, and gradient-enhanced HMBC. Data were analyzed and simulations performed using Varian VNMR software, version 6.1A.

GC-MSAnalysis. Trimethylsilyl-derivatized carbohydrates from buckwheat seeds containing Fagopyritol A1 and Fagopyritol B1 were chromatographed on a DB-5 capillary column (30 m×0.25 mm i.d., 0.25 μm film thickness) in a Hewlett Packard 5890 gas chromatograph coupled to a HP 5970 mass spectrometer. The temperature program was 140° C. for 4 min, increased at 3° C. min⁻¹ to 270° C., and held at 270° C. for 20 min. The carrier gas was helium at 1 mL/min. Mass spectra for GC peaks corresponding to derivatized Fagopyritol Al and Fagopyritol B1 were compared to mass spectra for trimethylsilyl derivatives of authentic D-chiro-inositol and D-galactose.

Results. Buckwheat bran was extracted with 50% EtOH, and the resulting extract chromatographed on carbon-Celite, 1:1 (w/w), to purify Fagopyritol A1. Fagopyritol B1 was also purified from the same sample for comparison. Per(pentafluoropropionates) of chiro-inositol from acid hydrolysis of Fagopyritol A1 and Fagopyritol B1 co-chromatographed on a chiral column with authentic D-chiro-inositol, but not with authentic L-chiro-inositol. Hydrolysis of Fagopyritol A1 and Fagopyritol B1 produced D-galactose and D-chiro-inositol in mole ratios of 1.01:1.00 for Fagopyritol A1 and 1.03:1.00 for Fagopyritol B1. Free D-chiro-inositol was obtained by acid hydrolysis of a mixture of Fagopyritol A1 and Fagopyritol B1 followed by purification on a carbon-Celite column. Fagopyritol A1 and Fagopyritol B1 were hydrolyzed by α-D-galactosidase, but not by β-D-galactosidase, demonstrating an α-D-galactopyranosyl linkage.

¹³C NMR spectra for Fagopyritol A1 identified 12 carbon resonances, and ¹H NMR spectra identified 13¹H resonances. The chemical shifts for the 12 carbons in Fagopyritol A1 were each linked to those of the 13 hydrogens using GHMQC. The characteristically downfield anomeric carbon (δ 100.26) and anomeric ¹H (δ 5.16) of the D-galactopyranosyl residue served as the starting point for the analysis of the DQCOSY for connectivity within the spin systems. The two coincident H-6′ hydrogens on the galactosyl residue were also a useful marker, clearly identifiable by a doublet with double intensity at δ 3.55 in the ¹H spectrum, and attached to the most upfield carbon (δ 61.80) in the ¹³C spectrum. The remaining resonance assignments for the galactosyl ring were straightforward. Once the ¹H signals for the galactose residue were assigned (Table 19), the six remaining resonances could be assigned to the D-chiro-inositol ring.

TABLE 19 ¹H and ¹³C NMR chemical shifts of Fagopyritol A1, Fagopyritol B1, and free D-chiro-inositol in D₂O at 25° C. Chemical Shifts (ppm) Fagopyritol free D- Fagopyritol A1 B1 chiro-inositol Position (no.) δ_(H) δ_(C) δ_(H) δ_(C) δ_(H) δ_(C) Cyclitol ring 1 3.85  72.56 4.04 68.46 3.85 72.43 2 3.67  69.51 3.64 76.61 3.58 71.22 3 3.54  81.77 3.53 72.04 3.41 73.52 4 3.61  73.61^(a) 3.44 73.48 3.41 73.52 5 3.61  71.06^(a) 3.60 71.19 3.58 71.22 6 3.83  72.08 3.87 71.85 3.85 72.43 Galactopyranosyl ring 1′ 5.16 100.26 4.93 96.42 2′ 3.66  69.87 3.67 69.06 3′ 3.73  70.19 3.77 70.10 4′ 3.81  69.96 3.83 69.93 5′ 4.07  71.86 4.03 71.63 6′a 3.55  61.80 3.55 61.69 6′b 3.55 3.55 ^(a)Assignment may be reversed

The hydrogen at 3.54 ppm was attached to the downfield carbon (δ 81.77) with a resonance shifted 8-10 ppm downfield in comparison to resonances for free D-chiro-inositol (Table 19), thereby identifying the point of galactose substitution on the D-chiro-inositol ring. Additionally, GHMBC and NOESY experiments showed clear interactions between the hydrogen at 3.54 and the anomeric hydrogen (δ 5.16; H-1′) and anomeric carbon (δ 100.26; C-1′) of galactose, confirming the point of linkage.

Coupling constants between adjacent hydrogens were calculated from the 1D ¹H signals. HOM2DJ aided in separating the multiplets in the region 3.5-3.9 ppm, which were formed from overlaid signals. The derived coupling constants (Table 20) allowed confirmation of the assignments made for the galactose ring of Fagopyritol A1.

TABLE 20 Estimated coupling constants derived from ¹H-NMR spectral data of Fagopyritol A1, Fagopyritol B1, and free D-chior-inositol Estimated proton-proton coupling constants (Hz) D-chiro- Fagopyritol A1 Fagopyritol B1 inositol Cyclitol Galactose Cyclitol Galactose Cyclitol J_(1,2) 3.2 J_(1′,2′)  3.9 J_(1,2) 2.9 J_(1′,2′)  3.9 J_(1,2) 2.6 J_(2,3) 9.7 J_(2′,3′) 10.4 J_(2,3) 9.7 J_(2′,3′) 10.4 J_(2,3) 9.8 J_(3,4) 9.7 J_(3′,4′)  3.2 J_(3,4) 9.6 J_(3′,4′)  3.4 J_(3,4) 9.8 J_(4,5) 9.7^(a) J_(4′,5′)  1.1 J_(4,5) 9.7 J_(4′,5′)  1.1 J_(4,5) 9.8 J_(5,6) 3.2 J_(5′,6′)  6.3 J_(5,6) 3.3 J_(5′,6′)  6.3 J_(5,6) 2.6 J_(6,1) 4.0 J_(6,1) 3.9 J_(6,1) 3.7 ^(a)Expected value; confirmed by simulation of experimental data

In particular, the small coupling constant J_(1′,2′) 3.9 Hz verified the α-anomeric linkage. The hydrogen at δ 3.54 on the linkage point of the D-chiro-inositol ring showed coupling constants of 9.7 Hz, indicating a trans-diaxial relationship with both adjacent hydrogens. Therefore, Fagopyritol A1 was assigned the structure O-α-D-galactopyranosyl-(1→3)-D-chiro-inositol. Fagopyritol A1 is isosteric with O-(2-amino-2-deoxy)-α-D-galactopyranosyl-(1→3)-D-chiro-inositol (Berlin et al., Tetrahedron Lett., 31:1109-1112 (1990), which is hereby incorporated by reference) related to a putative insulin mediator (Larner et al., Biochem. Biophys. Res. Comm., 151:1416-1426 (1988); Berlin et al., Tetrahedron Lett., 31:1109-1112 (1990), which are hereby incorporated by reference).

Coupling constants between adjacent hydrogens on the D-chiro-inositol ring were important in numbering the ring. However, the coupling constants for H-4 and H-5 of Fagopyritol A1 were not easily discernible. The intense interaction of these two strongly coupled hydrogens with coincident chemical shift (δ 3.61; H-4 and H-5), produced a complex multiplet with a maximum apparent splitting of 6.8 Hz. The three pairs of vicinal trans-diaxial hydrogens (H-2, H-3, H-4, H-5) in D-chiro-inositol should display coupling constants of 9-10 Hz, as observed for D-chiro-inositol in O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol (Fagopyritol B1, Table 20) (Szczecinski et al., Bull. Pol. Acad. Sci. Chem., 46:9-13 (1998), which is hereby incorporated by reference). The contradiction was resolved by simulation of the ¹H spectrum for the C-3 substituted D-chiro-inositol, using chemical shifts (Table 19) and coupling constants (Table 20) calculated from experimental spectral line data, but with the expected value of 9.7 Hz for J_(4,5). The simulated and experimental ¹H spectra were identical, confirming that the effect of the strong interaction between H-4 and H-5 resulted in a multiplet at 3.61 ppm with a maximum apparent splitting of 6.8 Hz. Thus, J_(4,5) is given as 9.7 Hz (Table 20) based on simulation. Assignment of the D-chiro-inositol ring hydrogens was easier for Fagopyritol B1 than for Fagopyritol A1 because the galactosyl substitution at H-3 of Fagopyritol A1 caused the adjacent H-4 of Fagopyritol A1 (δ 3.61) to be shifted downfield from that in Fagopyritol B1 (δ 3.44) and D-chiro-inositol (δ 3.41) such that it coincided with H-5 (δ 3.61). In Fagopyritol B1, H-4 (δ 3.44) was upfield of H-5 (δ 3.60) allowing the large coupling constant (J_(4,5) 9.7 Hz) of a trans-diaxial interaction to be observed for Fagopyritol B1 (Table 20). Simulation was also useful in the elucidation of coupling between hydrogens in free D-chiro-inositol. The ¹H NMR spectrum for D-chiro-inositol showed three multiplet patterns, each multiplet representing two identical hydrogens, two pairs of which had intra-pair coupling. Simulation of calculated chemical shifts and coupling constants gave a spectrum consistent with the observed spectrum for D-chiro-inositol and confirmed the calculated coupling constants as shown in Table 20.

Fagopyritol A1 and Fagopyritol B1 are positional isomers representing two different series of fagopyritol oligomers. ¹³C and/or ¹H resonances for Fagopyritol B1, O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol, were in close agreement with those reported for this structure in buckwheat (Szczecinski et al., Bull. Pol Acad. Sci. Chem., 46:9-13 (1998), which is hereby incorporated by reference), soybean (Schweizer et al., Carbohydr. Res., 95:61-71 (1981), which is hereby incorporated by reference), and jojoba bean (Ogawa et al., Carbohydr. Res., 302:219-221 (1997), which is hereby incorporated by reference). Resonances of the galactopyranosyl ring were in close agreement also with those of galactosylononitol (Richter et al., J. Nat. Prod., 60:749-751 (1997), which is hereby incorporated by reference) and other disaccharides containing galactose α-substituted at the anomeric carbon (Bock et al., Adv. Carbohydr. Chem. Biochem., 42:193-225 (1984), which is hereby incorporated by reference).

Mass spectra for trimethylsilylated products of Fagopyritol A1 and Fagopyritol B1 were similar with ion fragment peaks at m/z 73, 103, 129, 147, 191, 204, 217, 305, 318, 319, and 361. Abundance ratios of fragments with m/z 305/318 were 0.9 for Fagopyritol A1 and 2.6 for Fagopyritol B1, while abundance ratios of fragments with m/z 3181319 were 2.2 for Fagopyritol A1 and 0.9 for Fagopyritol B1. These abundance ratios may distinguish trimethylsilyl derivatives of Fagopyritol A1 and Fagopyritol B1 by GC-MS.

Example 24 Isolation and Characterization of Fagopyritol B3

Extraction ofFagopyritolsfrom Buckwheat. The bran milling fraction of common buckwheat (Fagopyrum esculentum Moench cv. Manor) seed as provided by Minn-Dak Growers, Ltd. (Grand Forks, N.Dak.). Buckwheat bran was extracted at pilot plant scale (see Example 21) with EtOH:H₂O 1:1 (v/v), the extract was filtered through a diatomaceous earth plate filter, evaporated to reduce the volume and remove EtOH, and treated with polyvinylpolypyrrolidone (PVPP), bentonite, and ion exchange resin to remove non-carbohydrate components. The soluble carbohydrate extract was finally filtered through 10,000 MW cut-off hollow fiber filter cartridges and evaporated to reduce the volume.

Purification ofFagopyritols. Purification was performed by preparative chromatography on a stationary phase of carbon (Darco G60; J. T. Baker, Phillipsburg, N.J.) and Celite 545-AW (Supelco, Bellefonte, Pa.), 1:1 (w/w) (Whistler and Durso, 1950). A mixture of 320 g of carbon and 320 g of Celite were mixed with water to a thin paste and placed into a 100 mm×300 mm column to a 200 mm bed depth (1570 mL bed volume). The column was loaded with 220 of pilot plant extract containing about 20 g of total sugars. Soluble carbohydrates were eluted stepwise at 4° C. with 4 L of glass distilled water, 4 L of 2% ethanol, 4 L of 4% ethanol, 4 L of 5% ethanol, 4 L of 8% ethanol, 4 L of 10% ethanol, 4 L of 14% ethanol, 2 L of 20% ethanol, and 2 L of 50% ethanol, and manually collected in 500-mL fractions. Flow rates ranged from 500 mL/h with water to about 175 mL/h with 50% ethanol. Volumes (rates) were too large for the fraction collectors, necessitating manual collection of fractions. Samples (200 μL) of each fraction were taken, phenyl α-D-glucoside (25 μg) was added as internal standard, dried samples were derivatized with N-trimethylsilylimidazole:pyridine, 1:1 (v/v), and analyzed (Horbowicz, which is hereby incorporated by reference) on a Hewlett Packard 6890 GC equipped with a flame ionization detector, split-mode injector (1:50), and an HP-1MS capillary column (15 m×0.25 mm i.d., 0.25 μm film thickness). The GC was operated with a programmed initial temperature of 150° C., adjusted to 200° C. at 3° C./min, adjusted to 325° C. at 7° C./min, and held at 325° C. for 20 min. The injector port was operated at 335° C. and the detector at 350° C. The carrier gas was nitrogen at 2.5 mL/min.

Results. Two columns were run (cc37 and cc38 (run with a refined elution protocol, as outlined above)). The elution sequence profile for column cc38 is illustrated in FIG. 16. D-chiro-inositol (chiro) and myo-inositol (myo) were eluted with water, fructose (Fru) and glucose (Glc) with 2% ethanol, Fagopyritol B1 with 4% ethanol, Fagopyritol A1 with 5% ethanol, excess sucrose with 8% ethanol, Fagopyritol B2 followed by DGMI with 10% ethanol, Fagopyritol A2 with 14% ethanol, Fagopyritol B3 and TGMI followed by Fagopyritol A3 with 20% ethanol and 50% ethanol. The 50% ethanol elution helps to concentrate the Fagopyritol A3 fraction. A peak tentatively identified as Fagopyritol B3 eluted before Fagopyritol A3 (FIG. 16).

Fagopyritol B3 eluted before Fagopyritol A3 as expected. Another compound tentatively identified as tri-galactosyl myo-inositol (TGMI; marked “T” in FIG. 17) eluted on the tail Fagopyritol B3, just as di-galactosyl myo-inositol (DGMI) eluted on the tail of Fagopyritol B2.

Fractions (from FIG. 16) containing fagopyritols were freeze dried. Fractions with similar contaminants were pooled and concentrated for further purification by additional carbon-Celite column chromatography. From column cc37 (not shown), two fractions (60 and 61) containing peaks identified as Fagopyritol B3 and TGMI were freeze dried, pooled, concentrated, and analyzed by high resolution gas chromatography. Data from a gas chromatogram of this concentrated fraction (60+61) was re-plotted in FIG. 18 with an expanded scale to emphasize the peaks of interest. Fagopyritol A3 had a retention time 3 8.27 min (A3), Fagopyritol B3 had a retention time 39.01 min (B3), and tri-galactosyl myo-inositol has a retention time of 39.46 min (TGMI) (FIG. 18; Table 21).

TABLE 21 Retention times for TMSI-derivatized sugars on typical GC chromatogram*. Retention time (min) Identification Front column Back column fructose (3 peaks) 4.44 4.28 4.64 4.48 4.77 4.61 glucose (2 peaks) 5.91 5.72 7.92 7.69 D-chiro-inositol 7.32 7.11 myo-inositol 9.97 9.71 phenyl α-D-glucoside 13.41 13.10 (internal standard) sucrose 21.46 21.26 Fagopyritol A1 24.19 23.99 Fagopyritol B1 24.75 24.55 galactinol 25.19 25.00 raffinose 28.97 28.78 Fagopyritol A2 31.79 31.58 Fagopyritol B2 32.23 32.02 di-galactosyl myo- 32.73 32.51 inositol Stachyose 35.34 35.07 Fagopyritol A3 38.27 37.82 Fagopyritol B3 39.01 38.49 tri-galactosyl myo- 39.46 38.95 inositol *Dried samples were derivatized with N-trimethylsilylimidazole:pyridine, 1:1 (v/v), and analyzed on a Hewlett at 3° C./min, adjusted to 325° C. at 7° C./min, and held at 325° C. for 20 min. The injector port was operated at 335° C. and the detector at 350° C. The carrier gas was nitrogen at 2.5 mL/min.

This chromatogram was run on the “back” column of the GC. If run on the “front” column, retention times were about 0.3 min longer.

Fagopyritol B3, in a mixture of fagopyritols, was completely hydrolyzed by α-galactosidase. A fraction enriched in Fagopyritol B3, but also containing Fagopyritol A3 and tri-galactosyl myo-inositol (TGMI), was completely hydrolyzed by trifluoroacetic acid to galactose and cyclitol. The mole ratio of galactose to cyclitol was 3:1. Analysis of the products after partial hydrolysis identified the presence of Fagopyritol B2 (O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol) and Fagopyritol B1 (O-α-(a-D-galactopyranosyl-(1→2)-D-chiro-inositol) (Fagopyritol B2 and Fagopyritol B1 were not present in the unhydrolyzed sample), thereby confirming the identity of Fagopyritol B3 as O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→2)-D-chiro-inositol.

Example 25 Isolation and Characterization of Fagopyritol A and B Series

Two complete series of buckwheat fagopyritols have been identified and characterized. The fagopyritol A series consists of Fagopyritol A1, Fagopyritol A2, and Fagopyritol A3. The fagopyritol B series consists of Fagopyritol B1, Fagopyritol B2 and Fagopyritol B3. All are hydrolyzed by α-galactosidase, but not by β-galactosidase, confirming that all fagopyritols are α-galactosides (Table 22).

TABLE 22 Hydrolysis products of Fagopyritol A Series and Fagopyritol B Series oligogalactosides Hydrolysis by 2 N trifluoroacetic acid Gal:cyclitol Partial hydrolysis Enzymic hydrolysis Soluble carbohydrate (mole ratio) products D-chiro-inositol L-chiro-inositol α-D-galactosidase β-D-galactosidase fagopyritol A Series Gal:D-chiro-inositol Fagopyritol A1 1.01:1.00 D-galactose yes no yes no D-chiro-inositol Fagopyritol A2 2.03:1.00 Fagopyritol A1 yes no yes no galactobiose D-galactose D-chiro-inositol Fagopyritol A3 2.98:1.00 Fagopyritol A2 yes no yes no Fagopyritol A1 galactotriose galactobiose D-galactose D-chiro-inositol fagopyritol B Series Gal:D-chiro-inositol Fagopyritol B1 1.03:1.00 D-galactose yes no yes no D-chiro-inositol Fagopyritol B2 2.02:1.00 Fagopyritol A1 yes no yes no galactobiose D-galactose D-chiro-inositol Fagopyritol B3   3:1 Fagopyritol B1 yes Fagopyritol B2

All are completely hydrolyzed to galactose and D-chiro-inositol by trifluoroacetic acid. Analysis of the acid hydrolysis products on a chiral capillary column by high resolution gas chromatography confirmed the presence of D-chiro-inositol in fagopyritols (Table 22). The mole ratio of galactose to D-chiro-inositol was 1:1 for Fagopyritol A1 and Fagopyritol B1, 2:1 for Fagopyritol A2 and Fagopyritol B2, and 3:1 for Fagopyritol A3 and Fagopyritol B3 (Table 22). The linkage and absolute configuration of Fagopyritol A1, Fagopyritol B1, and D-chiro-inositol were determined by NMR and of Fagopyritol B2, Fagopyritol A2, and Fagopyritol A3. Fagopyritol A1 is isosteric with a putative insulin mediator. Example 24 illustrates the isolation and identification of Fagopyritol B3. Fagopyritol B3 is a minor component in buckwheat seeds. The quantitative ratio of Fagopyritol A3 to Fagopyritol B3 is about 10:1.

Example 26-Evidence for the Identification of DGMI and TGMI in the Galactinol Series of Oligogalactosides.

Galactinol is O-α-D-galactopyranosyl-(1→1)-L-myo-inositol, an important galactosyl donor to the biosynthesis of the raffinose series oligosaccharides (raffinose, stachyose, verbascose), galactopinitols, and possibly fagopyritols. Members of the Galactinol Series have been proposed to be galactinol, di-galactosyl myo-inositol, tri-galactosyl myo-inositol, and tetra-galactosyl myo-inositol (Horbowicz and Petek 1, which are hereby incorporated by reference). The absolute configuration of galactinol has been determined by NMR. DGMI has not been analyzed by NMR. The tri- and tetra-galactosyl members of the galactinol series have been proposed, but not previously isolated or characterized.

Buckwheat seeds contain galactinol, di-galactosyl myo-inositol (DGMI), and tri-galactosyl myo-inositol (TGMI). DGMI elutes with 10% ethanol during carbon-Celite chromatography between Fagopyritol B2 and Fagopyritol A2 (see Example 24). During gas chromatography on a HP-1 MS capillary column, the peak for DGMI had a retention time about 0.5 min longer than the peak for Fagopyritol B2 (see Table 21). DGMI was completely hydrolyzed by α-galactosidase, but not by β-galactosidase, confirming that DGMI is an α-galactoside (Table 23).

TABLE 23 Hydrolysis products of the Galactinol Series oligogalactosides Hydrolysis by 2 N trifluoroacetic acid Partial Enzymic hydrolysis Soluble Gal:cyclitol hydrolysis α-D- β-D- carbohydrate (mole ratio) products galactosidase galactosidase Galactinol Gal:myo- Series inositol Galactinol (gtl) 1.03:1.00 D-galactose yes no myo-inositol Di-galactosyl 2.08:1.00 galactinol yes no myo-inositol galactobiose (DGMI) D-galactose myo-inositol Tri-galactosyl   3:1 galactinol yes myo-inositol myo-inositol (TGMI)

DGMI was completely hydrolyzed to galactose and myo-inositol in a mole ratio of 2.08:1.00 (Table 23). After partial hydrolysis, products identified were galactinol, galactobiose (gal-gal), D-galactose, and myo-inositol. These results confirmed the structure of DGMI as O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→1)-L-myo-inositol (also known as O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→3)-D-myo-inositol).

Tri-galactosyl myo-inositol (TGMI) elutes with 20% ethanol during carbon Celite chromatography between Fagopyritol B3 and Fagopyritol A3 (see Example 24). During gas chromatography on a HP-1MS capillary column, the peak for TGMI has a retention time about 0.45 min longer than the peak for Fagopyritol B3 (Table 21; FIG. 19). TGMI was completely hydrolyzed by α-galactosidase and by trifluoroacetic acid. The mole ratio of galactose to cyclitol was 1:3. During analysis of products after partial hydrolysis of a fraction enriched in TGMI and also containing Fagopyritol B3 and Fagopyritol A3 (but not galactinol, DGMI, or myo-inositol), both galactinol and myo-inositol were detected (Table 23). These results identify tri-galactosyl myo-inositol as O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→1)-L-myo-inositol (also known as O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→3)-D-myo-inositol).

Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims. 

What is claimed:
 1. An isolated fagopyritol selected from the group consisting of Fagopyritol A1, Fagopyritol A3, and Fagopyritol B3.
 2. An isolated fagopyritol according to claim 1, wherein said fagopyritol is Fagopyritol A1 having the formula:


3. An isolated fagopyritol according to claim 1, wherein said fagopyritol is Fagopyritol A3 having the formula:


4. An isolated fagopyritol according to claim 1, wherein said fagopyritol is Fagopyritol B3 having the formula:


5. An isolated fagopyritol according to claim 1, wherein said fagopyritol substantially free of one or more of galactinol, myo-inositol, digalactosyl myo-inositol, phytin, aromatic materials, cell wall particles, proteins, organic acids, amino acids, nucleic acids, and salts thereof.
 6. A composition comprised of two or more of Fagopyritol A1, Fagopyritol A2, Fagopyritol A3, Fagopyritol B1, Fagopyritol B2, Fagopyritol B3, and D-chiro-inositol, wherein said composition comprises at least one of the isolated fagopyritols according to claim
 1. 7. A composition according to claim 6, wherein the composition is substantially free of one or more of galactinol, myo-inositol, digalactosyl myo-inositol, phytin, aromatic materials, cell wall particles, proteins, organic acids, amino acids, nucleic acids, and salts thereof.
 8. A method of treating type II diabetes in a patient comprising: administering to the patient a composition according to claim 6 in an effective amount.
 9. A pharmaceutical composition for use in treating type II diabetes comprising: a pharmaceutical carrier and one or more isolated fagopyritols according to claim
 1. 10. A pharmaceutical composition according to claim 9, wherein said pharmaceutical composition further comprises a fagopyritol selected from the group consisting of Fagopyritol A2, Fagopyritol B1, Fagopyritol B2, and combinations thereof.
 11. A pharmaceutical composition according to claim 9, wherein said pharmaceutical composition consists essentially of said pharmaceutical carrier and said one or more isolated fagopyritols according to claim
 1. 12. A method of treating type II diabetes in a patient comprising: administering to the patient a pharmaceutical composition according to claim 9 in an effective amount.
 13. A method of treating type II diabetes in a patient comprising: administering to the patient one or more isolated fagopyritols according to claim 1 in an effective amount.
 14. A substantially pure fagopyritol selected from the group consisting of Fagopyritol A1, Fagopyritol A3, and Fagopyritol B3.
 15. A substantially pure fagopyritol according to claim 14, wherein said fagopyritol is Fagopyritol A1.
 16. A substantially pure fagopyritol according to claim 14, wherein said fagopyritol is Fagopyritol A3.
 17. A substantially pure fagopyritol according to claim 14, wherein said fagopyritol is Fagopyritol B3.
 18. A method of treating type II diabetes in a patient comprising: administering to the patient a substantially pure fagopyritol according to claim 14 in an effective amount.
 19. A method for preparing Fagopyritol A1, Fagopyritol A3, Fagopyritol B3, or a mixture thereof comprising: contacting buckwheat with a solvent under conditions effective to produce a crude extract comprising non-fagopyritol materials and one or more fagopyritols selected from the group consisting of Fagopyritol A1, Fagopyritol A3, and Fagopyritol B3; and separating the non-fagopyritol materials from the one or more fagopyritols.
 20. A method according to claim 19, wherein the buckwheat comprises buckwheat seed embryos.
 21. A method according to claim 19, wherein the buckwheat is buckwheat flour.
 22. A method according to claim 19, wherein the non-fagopyritol materials comprise one or more of cell wall particles, large proteins, charged materials, and aromatic materials.
 23. A method according to claim 19, wherein said separating comprises: evaporating the solvent under conditions effective to condense the crude extract.
 24. A method according to claim 19, wherein said separating comprises: passing the crude extract through a molecular weight cut off filter under conditions effective to remove cell wall particles and large proteins from the crude extract.
 25. A method according to claim 19, wherein said separating comprises: passing the crude extract through an ion exchange material under conditions effective to remove charged materials from the crude extract.
 26. A method according to claim 19, wherein said separating comprises: passing the crude extract through a polyvinylpyrrolidone column or an activated charcoal column under conditions effective to remove aromatic materials from the crude extract.
 27. A method according to claim 19 wherein the one or more fagopyritols comprises at least two fagopyritols, and wherein said method further comprises: separating one of Fagopyritol A1, Fagopyritol A3, and Fagopyritol B3 from the at least two fagopyritols.
 28. A method according to claim 27, wherein said separating one of Fagopyritol A1, Fagopyritol A3, and Fagopyritol B3 from the at least two fagopyritols is carried out chromatographically.
 29. A method according to claim 28, wherein said separating one of Fagopyritol A1, Fagopyritol A3, and Fagopyritol B3 from the at least two fagopyritols is carried out on a amine-functionalized silica gel.
 30. A method according to claim 28, wherein said separating one of Fagopyritol A1, Fagopyritol A3, and Fagopyritol B3 from the at least two fagopyritols is carried out using an acetonitrile: water eluent. 